Publications

@article{Rangel2022,

title = {Venom characterization of the Brazilian Pampa snake Bothrops pubescens by top-down and bottom-up proteomics},

author = {Rangel, D.L and Melani, R.D and Carvalho, E.L and Boldo, J.T and Gomes, Dos Santos T and Kelleher, N.L and Pinto, P.M

},

doi = {10.1016/j.toxicon.2022.106937},

year  = {2022},

date = {2022-12-10},

journal = {Toxicon},

abstract = {The envenomation from the Bothrops genus is characterized by systemic and local effects caused by the main toxin families in the venom. In Bothrops pubescens venom we were able to identify 89 protein groups belonging to 13 toxin families with the bottom-up proteomics approach and 40 unique proteoforms belonging to 6 toxin families with the top-down proteomics approach. We also identified multi-proteoform complexes of dimeric L-amino acid oxidase using native top-down mass spectrometry.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Forte2022,

title = {Divergent Antibody Repertoires Found for Omicron versus Wuhan SARS-CoV-2 Strains Using Ig-MS},

author = {Forte, E and Des Soye, B.J and Melani, R.D and Hollas, M.A.R and Kafader, J.O and Sha, B.E and Schneider, J.R and Kelleher, N.L},

doi = {10.1021/acs.jproteome.2c00514},

year  = {2022},

date = {2022-11-07},

urldate = {2022-11-07},

journal = {J Proteome Res.},

abstract = {SARS-CoV-2 Omicron (B.1.1.529) and its subvariants are currently the most common variants of concern worldwide, featuring numerous mutations in the spike protein and elsewhere that collectively make Omicron variants more transmissible and more resistant to antibody-mediated neutralization provided by vaccination, previous infections, and monoclonal antibody therapies than their predecessors. We recently reported the creation and characterization of Ig-MS, a new mass spectrometry-based serology platform that can define the repertoire of antibodies against an antigen of interest at single proteoform resolution. Here, we applied Ig-MS to investigate the evolution of plasma antibody repertoires against the receptor-binding domain (RBD) of SARS-CoV-2 in response to the booster shot and natural viral infection. We also assessed the capacity for antibody repertoires generated in response to vaccination and/or infection with the Omicron variant to bind to both Wuhan- and Omicron-RBDs. Our results show that (1) the booster increases antibody titers against both Wuhan- and Omicron- RBDs and elicits an Omicron-specific response and (2) vaccination and infection act synergistically in generating anti-RBD antibody repertoires able to bind both Wuhan- and Omicron-RBDs with variant-specific antibodies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Schachner2022,

title = {Revving an Engine of Human Metabolism: Activity Enhancement of Triosephosphate Isomerase via Hemi-Phosphorylation},

author = {Schachner, L.F and Soye, B.D and Ro, S and Kenney, G.E and Ives, A.N and Su,T and Goo, Y.A and Jewett, M.C and Rosenzweig, A.C and Kelleher, N.L},

doi = {10.1021/acschembio.2c00324},

year  = {2022},

date = {2022-10-21},

urldate = {2022-10-21},

journal = {ACS Chem Biol.},

volume = {17},

number = {10},

pages = {2769-2780},

abstract = {Triosephosphate isomerase (TPI) performs the 5th step in glycolysis, operates near the limit of diffusion, and is involved in "moonlighting" functions. Its dimer was found singly phosphorylated at Ser20 (pSer20) in human cells, with this post-translational modification (PTM) showing context-dependent stoichiometry and loss under oxidative stress. We generated synthetic pSer20 proteoforms using cell-free protein synthesis that showed enhanced TPI activity by 4-fold relative to unmodified TPI. Molecular dynamics simulations show that the phosphorylation enables a channel to form that shuttles substrate into the active site. Refolding, kinetic, and crystallographic analyses of point mutants including S20E/G/Q indicate that hetero-dimerization and subunit asymmetry are key features of TPI. Moreover, characterization of an endogenous human TPI tetramer also implicates tetramerization in enzymatic regulation. S20 is highly conserved across eukaryotic TPI, yet most prokaryotes contain E/D at this site, suggesting that phosphorylation of human TPI evolved a new switch to optionally boost an already fast enzyme. Overall, complete characterization of TPI shows how endogenous proteoform discovery can prioritize functional versus bystander PTMs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Aoi2022,

title = {SPT6 functions in transcriptional pause/release via PAF1C recruitment},

author = {Aoi, Y and Shah, A.P and Ganesan, S and Soliman, S.H.A and Cho, B.K and Goo, Y.A and Kelleher, N.L and Shilatifard, A},

url = {https://pubmed.ncbi.nlm.nih.gov/35973425/},

doi = {10.1016/j.molcel.2022.06.037},

year  = {2022},

date = {2022-09-15},

journal = {Mol Cell.},

volume = {82},

number = {18},

pages = {3412-3423.e5},

abstract = {It is unclear how various factors functioning in the transcriptional elongation by RNA polymerase II (RNA Pol II) cooperatively regulate pause/release and productive elongation in living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in the release of paused RNA Pol II into gene bodies through an impaired recruitment of PAF1C. Short genes demonstrate a release with increased mature transcripts, whereas long genes are released but fail to yield mature transcripts, due to a reduced processivity resulting from both SPT6 and PAF1C loss. Unexpectedly, SPT6 depletion causes an association of NELF with the elongating RNA Pol II on gene bodies, without any observed functional significance on transcriptional elongation pattern, arguing against a role for NELF in keeping RNA Pol II in the paused state. Furthermore, SPT6 depletion impairs heat-shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through PAF1C recruitment.



},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Charna2022,

title = {An efficient cell-free protein synthesis platform for producing proteins with pyrrolysine-based noncanonical amino acids},

author = {Ranji Charna, A and Des Soye, B.J and Ntai, I and Kelleher, N.L and Jewett, M.C

},

doi = {10.1002/biot.202200096},

year  = {2022},

date = {2022-09-01},

journal = {Biotechnol J.},

volume = {17},

number = {9},

abstract = {Incorporation of noncanonical amino acids (ncAAs) into proteins opens new opportunities in biotechnology and synthetic biology. Pyrrolysine (Pyl)-based ncAAs are some of the most predominantly used, but expression systems suffer from low yields. Here, we report a highly efficient cell-free protein synthesis (CFPS) platform for site-specific incorporation of Pyl-based ncAAs into proteins using amber suppression. This platform is based on cellular extracts derived from genomically recoded Escherichia coli lacking release factor 1 and enhanced through deletion of endonuclease A. To enable ncAA incorporation, orthogonal translation system (OTS) components (i.e., the orthogonal transfer RNA [tRNA] and orthogonal aminoacyl tRNA synthetase) were coexpressed in the source strain prior to lysis and the orthogonal tRNACUA Pyl that decodes the amber codon was further enriched in the CFPS reaction via co-synthesis with the product. Using this platform, we demonstrate production of up to 442 ± 23 µg/mL modified superfolder green fluorescent protein (sfGFP) containing a single Pyl-based ncAA at high (>95%) suppression efficiency, as well as sfGFP variants harboring multiple, identical ncAAs. Our CFPS platform can be used for the synthesis of modified proteins containing multiple precisely positioned, genetically encoded Pyl-based ncAAs. We anticipate that it will facilitate more general use of CFPS in synthetic biology.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Su2022,

title = {Highly multiplexed, label-free proteoform imaging of tissues by individual ion mass spectrometry},

author = {Su, P and McGee, J.P and Durbin, K.R and Hollas, M.A.R and Yang, M and Neumann, E.K and Allen, J.L and Drown, B.S and Butun, F.A and Greer, J.B and Early, B.P and Fellers, R.T and Spraggins, J.M and Laskin, J and Camarillo, J.M and Kafader, J.O and Kelleher, N.L},

doi = {10.1126/sciadv.abp9929},

year  = {2022},

date = {2022-08-12},

journal = {Sci Adv. },

volume = {8},

number = {32},

abstract = {Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by fourfold compared to reported methods and reveals tissue localization of proteoforms at <80-μm spatial resolution. PiMS advances proteoform imaging by combining ambient nanospray desorption electrospray ionization with ion detection using individual ion mass spectrometry. We demonstrate highly multiplexed proteoform imaging of human kidney, annotating 169 of 400 proteoforms of <70 kDa using top-down MS and a database lookup of ~1000 kidney candidate proteoforms, including dozens of key enzymes in primary metabolism. PiMS images reveal distinct spatial localizations of proteoforms to both anatomical structures and cellular neighborhoods in the vasculature, medulla, and cortex regions of the human kidney. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of tissues.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Jooß2022,

title = {Native Mass Spectrometry at the Convergence of Structural Biology and Compositional Proteomics},

author = {Jooß, K and McGee, J.P and Kelleher, N.L

},

doi = {10.1021/acs.accounts.2c00216},

year  = {2022},

date = {2022-07-19},

journal = {Acc Chem Res.},

volume = {55},

number = {14},

pages = {1928-1937},

abstract = {Biology is driven by a vast set of molecular interactions that evolved over billions of years. Just as covalent modifications like acetylations and phosphorylations can change a protein's function, so too can noncovalent interactions with metals, small molecules, and other proteins. However, much of the language of protein-level biology is left either undiscovered or inferred, as traditional methods used in the field of proteomics use conditions that dissociate noncovalent interactions and denature proteins.Just in the past few years, mass spectrometry (MS) has evolved the capacity to preserve and subsequently characterize the complete composition of endogenous protein complexes. Using this "native" type of mass spectrometry, a complex can be activated to liberate some or all of its subunits, typically via collisions with neutral gas or solid surfaces and isolated before further characterization ("Native Top-Down MS," or nTDMS). The subunit mass, the parent ion mass, and the fragment ions of the activated subunits can be used to piece together the precise molecular composition of the parent complex. When performed en masse in discovery mode (i.e., "native proteomics"), the interactions of life─including protein modifications─will eventually be clarified and linked to dysfunction in human disease states.In this Account, we describe the current and future components of the native MS toolkit, covering the challenges the field faces to characterize ever larger bioassemblies. Each of the three pillars of native proteomics are addressed: (i) separations, (ii) top-down mass spectrometry, and (iii) integration with structural biology. Complexes such as endogenous nucleosomes can be targeted now using nTDMS, whereas virus particles, exosomes, and high-density lipoprotein particles will be tackled in the coming few years. The future work to adequately address the size and complexity of mega- to gigadalton complexes will include native separations, single ion mass spectrometry, and new data types. The use of nTDMS in discovery mode will incorporate native-compatible separation techniques to maximize the number of proteoforms in complexes identified. With a new wave of innovations, both targeted and discovery mode nTDMS will expand to include extremely scarce and exceedingly heterogeneous bioassemblies. Understanding the proteinaceous interactions of life and how they go wrong (e.g., misfolding, forming complexes in dysfunctional stoichiometries and configurations) will not only inform the development of life-restoring therapeutics but also deepen our understanding of life at the molecular level.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Yang2022,

title = {Proteoform-Selective Imaging of Tissues Using Mass Spectrometry},

author = {Yang, M and Hu, H and Su, P and Thomas, P.M and Camarillo, J.M and Greer, J.B and Early, B.P and Fellers, R.T and Kelleher, N.L and Laskin, J

},

doi = {10.1002/anie.202200721},

year  = {2022},

date = {2022-07-18},

journal = {Angew Chem Int Ed Engl.},

volume = {61},

number = {29},

abstract = {Unraveling the complexity of biological systems relies on the development of new approaches for spatially resolved proteoform-specific analysis of the proteome. Herein, we employ nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI) for the proteoform-selective imaging of biological tissues. Nano-DESI generates multiply charged protein ions, which is advantageous for their structural characterization using tandem mass spectrometry (MS/MS) directly on the tissue. Proof-of-concept experiments demonstrate that nano-DESI MSI combined with on-tissue top-down proteomics is ideally suited for the proteoform-selective imaging of tissue sections. Using rat brain tissue as a model system, we provide the first evidence of differential proteoform expression in different regions of the brain.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Leung2022,

title = {SETD2 Haploinsufficiency Enhances Germinal Center-Associated AICDA Somatic Hypermutation to Drive B-cell Lymphomagenesis},

author = {Leung, W and Teater, M and Durmaz, C and Meydan, C and Chivu, A.G and Chadburn, A and Rice, E.J and Muley, A and Camarillo, J.M and Arivalagan, J and Li, Z and Flowers, C.R and Kelleher, N.L and Danko, C.G and Imielinski, M and Dave, S.S and Armstrong, S.A and Mason, C.E and Melnick, A.M},

doi = {10.1158/2159-8290.CD-21-1514},

year  = {2022},

date = {2022-07-06},

journal = {Cancer Discov.},

volume = {12},

number = {7},

pages = {1782-1803},

abstract = {SETD2 is the sole histone methyltransferase responsible for H3K36me3, with roles in splicing, transcription initiation, and DNA damage response. Homozygous disruption of SETD2 yields a tumor suppressor effect in various cancers. However, SETD2 mutation is typically heterozygous in diffuse large B-cell lymphomas. Here we show that heterozygous Setd2 deficiency results in germinal center (GC) hyperplasia and increased competitive fitness, with reduced DNA damage checkpoint activity and apoptosis, resulting in accelerated lymphomagenesis. Impaired DNA damage sensing in Setd2-haploinsufficient germinal center B (GCB) and lymphoma cells associated with increased AICDA-induced somatic hypermutation, complex structural variants, and increased translocations including those activating MYC. DNA damage was selectively increased on the nontemplate strand, and H3K36me3 loss was associated with greater RNAPII processivity and mutational burden, suggesting that SETD2-mediated H3K36me3 is required for proper sensing of cytosine deamination. Hence, Setd2 haploinsufficiency delineates a novel GCB context-specific oncogenic pathway involving defective epigenetic surveillance of AICDA-mediated effects on transcribed genes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Amici2022,

title = {C16orf72/HAPSTR1 is a molecular rheostat in an integrated network of stress response pathways},

author = {Amici, D.R and Ansel, D.J and Metz, K.A and Smith, R.S and Phoumyvong, C.M and Gayatri, S and Chamera, T and Edwards, S.L and O'Hara, B.P and Srivastava, S and Brockway, S and Takagishi, S.R and Cho, B.K and Goo, Y.A and Kelleher, N.L and Ben-Sahra, I and Foltz, D.R and Li, J and Mendillo, M.L

},

doi = {10.1073/pnas.2111262119},

year  = {2022},

date = {2022-07-05},

journal = {Proc Natl Acad Sci U S A.},

volume = {119},

number = {27},

abstract = {All cells contain specialized signaling pathways that enable adaptation to specific molecular stressors. Yet, whether these pathways are centrally regulated in complex physiological stress states remains unclear. Using genome-scale fitness screening data, we quantified the stress phenotype of 739 cancer cell lines, each representing a unique combination of intrinsic tumor stresses. Integrating dependency and stress perturbation transcriptomic data, we illuminated a network of genes with vital functions spanning diverse stress contexts. Analyses for central regulators of this network nominated C16orf72/HAPSTR1, an evolutionarily ancient gene critical for the fitness of cells reliant on multiple stress response pathways. We found that HAPSTR1 plays a pleiotropic role in cellular stress signaling, functioning to titrate various specialized cell-autonomous and paracrine stress response programs. This function, while dispensable to unstressed cells and nematodes, is essential for resilience in the presence of stressors ranging from DNA damage to starvation and proteotoxicity. Mechanistically, diverse stresses induce HAPSTR1, which encodes a protein expressed as two equally abundant isoforms. Perfectly conserved residues in a domain shared between HAPSTR1 isoforms mediate oligomerization and binding to the ubiquitin ligase HUWE1. We show that HUWE1 is a required cofactor for HAPSTR1 to control stress signaling and that, in turn, HUWE1 feeds back to ubiquitinate and destabilize HAPSTR1. Altogether, we propose that HAPSTR1 is a central rheostat in a network of pathways responsible for cellular adaptability, the modulation of which may have broad utility in human disease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Melani2022b,

title = {Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire},

author = {Melani, R.D and Des Soye, B.J and Kafader, J.O and Forte, E and Hollas, M and Blagojevic, V and Negrão, F and McGee, J.P and Drown, B and Lloyd-Jones, C and Seckler, H.S and Camarillo, J.M and Compton, P.D and LeDuc, R.D and Early, B and Fellers, R.T and Cho, B.K and Mattamana, B.B and Goo, Y.A and Thomas, P.M and Ash, M.K and Bhimalli, P.P and Al-Harthi, L and Sha, B.E and Schneider, J.R and Kelleher, N.L},

doi = {10.1021/acs.jproteome.1c00882},

year  = {2022},

date = {2022-07-01},

journal = {J Proteome Res.},

volume = {21},

number = {1},

pages = {274-288},

abstract = {Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multiparametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We applied Ig-MS to plasma from subjects with severe and mild COVID-19 and immunized subjects after two vaccine doses, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, that could use other antigens of interest to gauge immune responses to vaccination, pathogens, or autoimmune disorders.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Johnson2022,

title = {New Views of Old Proteins: Clarifying the Enigmatic Proteome},

author = {Burnum-Johnson, K.E and Conrads, T.P and Drake, R.R and Herr, A.E and Iyengar, R and Kelly, R.T and Lundberg, E and MacCoss, M.J and Naba, A and Nolan, G.P and Pevzner, P.A and Rodland, K.D and Sechi, S and Slavov, N and Spraggins, J.M and Van Eyk, J.E and Vidal, M and Vogel, C and Walt, D.R and Kelleher, N.L

},

doi = {10.1016/j.mcpro.2022.100254},

year  = {2022},

date = {2022-05-30},

journal = {Mol Cell Proteomics.},

volume = {21},

number = {7},

abstract = {All human diseases involve proteins, yet our current tools to characterize and quantify them are limited. To better elucidate proteins across space, time, and molecular composition, we provide a >10 years of projection for technologies to meet the challenges that protein biology presents. With a broad perspective, we discuss grand opportunities to transition the science of proteomics into a more propulsive enterprise. Extrapolating recent trends, we describe a next generation of approaches to define, quantify, and visualize the multiple dimensions of the proteome, thereby transforming our understanding and interactions with human disease in the coming decade.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Drown2022,

title = {Mapping the Proteoform Landscape of Five Human Tissues},

author = {Drown, B.S and Jooß, K and Melani, R.D and Lloyd-Jones, C and Camarillo, J.M and Kelleher, N.L

},

doi = {10.1021/acs.jproteome.2c00034},

year  = {2022},

date = {2022-05-06},

journal = {J Proteome Res.},

volume = {21},

number = {5},

pages = {1299-1310},

abstract = {A functional understanding of the human body requires structure-function studies of proteins at scale. The chemical structure of proteins is controlled at the transcriptional, translational, and post-translational levels, creating a variety of products with modulated functions within the cell. The term "proteoform" encapsulates this complexity at the level of chemical composition. Comprehensive mapping of the proteoform landscape in human tissues necessitates analytical techniques with increased sensitivity and depth of coverage. Here, we took a top-down proteomics approach, combining data generated using capillary zone electrophoresis (CZE) and nanoflow reversed-phase liquid chromatography (RPLC) hyphenated to mass spectrometry to identify and characterize proteoforms from the human lungs, heart, spleen, small intestine, and kidneys. CZE and RPLC provided complementary post-translational modification and proteoform selectivity, thereby enhancing the overall proteome coverage when used in combination. Of the 11,466 proteoforms identified in this study, 7373 (64%) were not reported previously. Large differences in the protein and proteoform level were readily quantified, with initial inferences about proteoform biology operative in the analyzed organs. Differential proteoform regulation of defensins, glutathione transferases, and sarcomeric proteins across tissues generate hypotheses about how they function and are regulated in human health and disease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{LeDuc2022,

title = {Proteomics Standards Initiative's ProForma 2.0: Unifying the Encoding of Proteoforms and Peptidoforms},

author = {LeDuc, R.D and Deutsch, E.W and Binz, P.A and Fellers, R.T and Cesnik, A.J and Klein, J.A and Van Den Bossche, T and Gabriels, R and Yalavarthi, A and Perez-Riverol, Y and Carver, J and Bittremieux, W and Kawano, S and Pullman, B and Bandeira, N and Kelleher, N.L and Thomas, P.M and Vizcaíno, J.A},

doi = {10.1021/acs.jproteome.1c00771},

year  = {2022},

date = {2022-04-01},

journal = {J Proteome Res.},

volume = {21},

number = {4},

pages = {1189-1195},

abstract = {It is important for the proteomics community to have a standardized manner to represent all possible variations of a protein or peptide primary sequence, including natural, chemically induced, and artifactual modifications. The Human Proteome Organization Proteomics Standards Initiative in collaboration with several members of the Consortium for Top-Down Proteomics (CTDP) has developed a standard notation called ProForma 2.0, which is a substantial extension of the original ProForma notation developed by the CTDP. ProForma 2.0 aims to unify the representation of proteoforms and peptidoforms. ProForma 2.0 supports use cases needed for bottom-up and middle-/top-down proteomics approaches and allows the encoding of highly modified proteins and peptides using a human- and machine-readable string. ProForma 2.0 can be used to represent protein modifications in a specified or ambiguous location, designated by mass shifts, chemical formulas, or controlled vocabulary terms, including cross-links (natural and chemical) and atomic isotopes. Notational conventions are based on public controlled vocabularies and ontologies. The most up-to-date full specification document and information about software implementations are available at http://psidev.info/proforma.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhu2022,

title = {Rational Design, Synthesis, and Mechanism of (3 S,4 R)-3-Amino-4-(difluoromethyl)cyclopent-1-ene-1-carboxylic Acid: Employing a Second-Deprotonation Strategy for Selectivity of Human Ornithine Aminotransferase over GABA Aminotransferase},

author = {Zhu, W and Butrin, A and Melani, R.D and Doubleday, P.F and Ferreira, G.M and Tavares, M.T and Habeeb Mohammad, T.S and Beaupre, B.A and Kelleher, N.L and Moran, G.R and Liu, D and Silverman, R.B},

doi = {10.1021/jacs.2c00924},

year  = {2022},

date = {2022-03-30},

journal = {J Am Chem Soc.},

volume = {144},

number = {12},

pages = {5629-5642},

abstract = {Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that contains a similar active site to that of γ-aminobutyric acid aminotransferase (GABA-AT). Recently, pharmacological inhibition of hOAT was recognized as a potential therapeutic approach for hepatocellular carcinoma. In this work, we first studied the inactivation mechanisms of hOAT by two well-known GABA-AT inactivators (CPP-115 and OV329). Inspired by the inactivation mechanistic difference between these two aminotransferases, a series of analogues were designed and synthesized, leading to the discovery of analogue 10b as a highly selective and potent hOAT inhibitor. Intact protein mass spectrometry, protein crystallography, and dialysis experiments indicated that 10b was converted to an irreversible tight-binding adduct (34) in the active site of hOAT, as was the unsaturated analogue (11). The comparison of kinetic studies between 10b and 11 suggested that the active intermediate (17b) was only generated in hOAT and not in GABA-AT. Molecular docking studies and pKa computational calculations highlighted the importance of chirality and the endocyclic double bond for inhibitory activity. The turnover mechanism of 10b was supported by mass spectrometric analysis of dissociable products and fluoride ion release experiments. Notably, the stopped-flow experiments were highly consistent with the proposed mechanism, suggesting a relatively slow hydrolysis rate for hOAT. The novel second-deprotonation mechanism of 10b contributes to its high potency and significantly enhanced selectivity for hOAT inhibition.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Park2022,

title = {A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis},

author = {Park, Y.J and Jodts, R.J and Slater, J.W and Reyes, R.M and Winton, V.J and Montaser, R.A and Thomas, P.M and Dowdle, W.B and Ruiz, A and Kelleher, N.L and Bollinger, J.M. Jr and Krebs, C and Hoffman, B.M and Rosenzweig, A.C

},

doi = {10.1073/pnas.2123566119},

year  = {2022},

date = {2022-03-29},

journal = {Proc Natl Acad Sci U S A.},

volume = {119},

number = {13},

abstract = {SignificanceMethanobactins (Mbns), copper-binding peptidic compounds produced by some bacteria, are candidate therapeutics for human diseases of copper overload. The paired oxazolone-thioamide bidentate ligands of methanobactins are generated from cysteine residues in a precursor peptide, MbnA, by the MbnBC enzyme complex. MbnBC activity depends on the presence of iron and oxygen, but the catalytically active form has not been identified. Here, we provide evidence that a dinuclear Fe(II)Fe(III) center in MbnB, which is the only representative of a >13,000-member protein family to be characterized, is responsible for this reaction. These findings expand the known roles of diiron enzymes in biology and set the stage for mechanistic understanding, and ultimately engineering, of the MbnBC biosynthetic complex.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Greer2022,

title = {ProSight Annotator: Complete control and customization of protein entries in UniProt XML files},

author = {Greer, J.B and Early, B.P and Durbin, K.R and Patrie, S.M and Thomas, P.M and Kelleher, N.L and LeDuc, R.D and Fellers, R.T},

doi = {10.1002/pmic.202100209},

year  = {2022},

date = {2022-03-23},

journal = {Proteomics.},

volume = {22},

number = {11-12},

abstract = {The effectiveness of any proteomics database search depends on the theoretical candidate information contained in the protein database. Unfortunately, candidate entries from protein databases such as UniProt rarely contain all the post-translational modifications (PTMs), disulfide bonds, or endogenous cleavages of interest to researchers. These omissions can limit discovery of novel and biologically important proteoforms. Conversely, searching for a specific proteoform becomes a computationally difficult task for heavily modified proteins. Both situations require updates to the database through user-annotated entries. Unfortunately, manually creating properly formatted UniProt Extensible Markup Language (XML) files is tedious and prone to errors. ProSight Annotator solves these issues by providing a graphical interface for adding user-defined features to UniProt-formatted XML files for better informed proteoform searches. It can be downloaded from http://prosightannotator.northwestern.edu.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Plubell2022,

title = {Putting Humpty Dumpty Back Together Again: What Does Protein Quantification Mean in Bottom-Up Proteomics?},

author = {Plubell, D.L and Käll, L and Webb-Robertson, B.J and Bramer, L.M and Ives, A and Kelleher, N.L and Smith, L.M and Montine, T.J and Wu, C.C and MacCoss, M.J},

doi = {10.1021/acs.jproteome.1c00894},

year  = {2022},

date = {2022-02-27},

journal = {J Proteome Res.},

volume = {21},

number = {4},

pages = {891-898},

abstract = {Bottom-up proteomics provides peptide measurements and has been invaluable for moving proteomics into large-scale analyses. Commonly, a single quantitative value is reported for each protein-coding gene by aggregating peptide quantities into protein groups following protein inference or parsimony. However, given the complexity of both RNA splicing and post-translational protein modification, it is overly simplistic to assume that all peptides that map to a singular protein-coding gene will demonstrate the same quantitative response. By assuming that all peptides from a protein-coding sequence are representative of the same protein, we may miss the discovery of important biological differences. To capture the contributions of existing proteoforms, we need to reconsider the practice of aggregating protein values to a single quantity per protein-coding gene.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Bartley2022,

title = {An Essential Role for Alzheimer's-Linked Amyloid Beta Oligomers in Neurodevelopment: Transient Expression of Multiple Proteoforms during Retina Histogenesis},

author = {Bartley, S.C and Proctor, M.T and Xia, H and Ho, E and Kang, D.S and Schuster, K and Bicca, M.A and Seckler, H.S and Viola, K.L and Patrie, S.M and Kelleher, N.L and De Mello, F.G and Klein, W.L},

doi = {10.3390/ijms23042208},

year  = {2022},

date = {2022-02-17},

journal = {Int J Mol Sci.},

volume = {23},

number = {4},

pages = {2208},

abstract = {Human amyloid beta peptide (Aβ) is a brain catabolite that at nanomolar concentrations can form neurotoxic oligomers (AβOs), which are known to accumulate in Alzheimer's disease. Because a predisposition to form neurotoxins seems surprising, we have investigated whether circumstances might exist where AβO accumulation may in fact be beneficial. Our investigation focused on the embryonic chick retina, which expresses the same Aβ as humans. Using conformation-selective antibodies, immunoblots, mass spectrometry, and fluorescence microscopy, we discovered that AβOs are indeed present in the developing retina, where multiple proteoforms are expressed in a highly regulated cell-specific manner. The expression of the AβO proteoforms was selectively associated with transiently expressed phosphorylated Tau (pTau) proteoforms that, like AβOs, are linked to Alzheimer's disease (AD). To test whether the AβOs were functional in development, embryos were cultured ex ovo and then injected intravitreally with either a beta-site APP-cleaving enzyme 1 (BACE-1) inhibitor or an AβO-selective antibody to prematurely lower the levels of AβOs. The consequence was disrupted histogenesis resulting in dysplasia resembling that seen in various retina pathologies. We suggest the hypothesis that embryonic AβOs are a new type of short-lived peptidergic hormone with a role in neural development. Such a role could help explain why a peptide that manifests deleterious gain-of-function activity when it oligomerizes in the aging brain has been evolutionarily conserved.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Melani2022,

title = {The Blood Proteoform Atlas: A reference map of proteoforms in human hematopoietic cells},

author = {Melani, R.D and Gerbasi, V.R and Anderson, L.C and Sikora, J.W and Toby, T.K and Hutton, J.E and Butcher, D.S and Negrão, F and Seckler, H.S and Srzentić, K and Fornelli, L and Camarillo, J.M and LeDuc, R.D and Cesnik, A.J and Lundberg, E and Greer, J.B and Fellers, R.T and Robey, M.T and DeHart, C.J and Forte, E and Hendrickson, C.L and Abbatiello, S.E and Thomas, P.M and Kokaji, A.I and Levitsky, J and Kelleher, N.L},

doi = {10.1126/science.aaz5284},

year  = {2022},

date = {2022-01-28},

journal = {Science.},

volume = {375},

number = {6579},

pages = {411-418},

abstract = {Human biology is tightly linked to proteins, yet most measurements do not precisely determine alternatively spliced sequences or posttranslational modifications. Here, we present the primary structures of ~30,000 unique proteoforms, nearly 10 times more than in previous studies, expressed from 1690 human genes across 21 cell types and plasma from human blood and bone marrow. The results, compiled in the Blood Proteoform Atlas (BPA), indicate that proteoforms better describe protein-level biology and are more specific indicators of differentiation than their corresponding proteins, which are more broadly expressed across cell types. We demonstrate the potential for clinical application, by interrogating the BPA in the context of liver transplantation and identifying cell and proteoform signatures that distinguish normal graft function from acute rejection and other causes of graft dysfunction.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hollas2022,

title = {The Human Proteoform Atlas: a FAIR community resource for experimentally derived proteoforms},

author = {Hollas, M.A.R and Robey, M.T and Fellers, R.T and LeDuc, R.D and Thomas, P.M and Kelleher, N.L},

doi = {0.1093/nar/gkab1086},

year  = {2022},

date = {2022-01-07},

journal = {Nucleic Acids Res.},

volume = {50},

number = {D1},

pages = {D526-D533},

abstract = {The Human Proteoform Atlas (HPfA) is a web-based repository of experimentally verified human proteoforms on-line at http://human-proteoform-atlas.org and is a direct descendant of the Consortium of Top-Down Proteomics' (CTDP) Proteoform Atlas. Proteoforms are the specific forms of protein molecules expressed by our cells and include the unique combination of post-translational modifications (PTMs), alternative splicing and other sources of variation deriving from a specific gene. The HPfA uses a FAIR system to assign persistent identifiers to proteoforms which allows for redundancy calling and tracking from prior and future studies in the growing community of proteoform biology and measurement. The HPfA is organized around open ontologies and enables flexible classification of proteoforms. To achieve this, a public registry of experimentally verified proteoforms was also created. Submission of new proteoforms can be processed through email vianrtdphelp@northwestern.edu, and future iterations of these proteoform atlases will help to organize and assign function to proteoforms, their PTMs and their complexes in the years ahead.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hickey2021,

title = {Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging},

author = {Hickey, J.W and Neumann, E.K and Radtke, A.J and Camarillo, J.M and Beuschel, R.T and Albanese, A and McDonough, E and Hatler, J and Wiblin, A.E and Fisher, J and Croteau, J and Small, E.C and Sood, A and Caprioli, R.M and Angelo, R.M and Nolan, G.P and Chung, K and Hewitt, S.M and Germain, R.N and Spraggins, J.M and Lundberg, E and Snyder, M.P and Kelleher, N.L and Saka, S.K},

doi = {10.1038/s41592-021-01316-y},

year  = {2021},

date = {2021-11-22},

urldate = {2021-11-22},

journal = {Nat Methods.},

volume = {19},

number = {3},

pages = {284-295},

abstract = {Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Caesar2021,

title = {Metabolomics and genomics in natural products research: complementary tools for targeting new chemical entities},

author = {Caesar, L.K and Montaser, R and Keller, N.P and Kelleher, N.L},

doi = {10.1039/d1np00036e},

year  = {2021},

date = {2021-11-17},

urldate = {2021-11-17},

journal = {Nat Prod Rep.},

volume = {38},

number = {11},

pages = {2041-2065},

abstract = {Covering: 2010 to 2021Organisms in nature have evolved into proficient synthetic chemists, utilizing specialized enzymatic machinery to biosynthesize an inspiring diversity of secondary metabolites. Often serving to boost competitive advantage for their producers, these secondary metabolites have widespread human impacts as antibiotics, anti-inflammatories, and antifungal drugs. The natural products discovery field has begun a shift away from traditional activity-guided approaches and is beginning to take advantage of increasingly available metabolomics and genomics datasets to explore undiscovered chemical space. Major strides have been made and now enable -omics-informed prioritization of chemical structures for discovery, including the prospect of confidently linking metabolites to their biosynthetic pathways. Over the last decade, more integrated strategies now provide researchers with pipelines for simultaneous identification of expressed secondary metabolites and their biosynthetic machinery. However, continuous collaboration by the natural products community will be required to optimize strategies for effective evaluation of natural product biosynthetic gene clusters to accelerate discovery efforts. Here, we provide an evaluative guide to scientific literature as it relates to studying natural product biosynthesis using genomics, metabolomics, and their integrated datasets. Particular emphasis is placed on the unique insights that can be gained from large-scale integrated strategies, and we provide source organism-specific considerations to evaluate the gaps in our current knowledge.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Smith2021,

title = {The Human Proteoform Project: Defining the human proteome},

author = {Smith, L.M and Agar, J.N and Chamot-Rooke, J and Danis, P.O and Ge, Y and Loo, J.A and Paša-Tolić, L and Tsybin, Y.O and Kelleher, N.L and Consortium for Top-Down Proteomics},

doi = {10.1126/sciadv.abk0734},

year  = {2021},

date = {2021-11-12},

urldate = {2021-11-12},

journal = {Sci Adv.},

volume = {7},

number = {46},

abstract = {Proteins are the primary effectors of function in biology, and thus, complete knowledge of their structure and properties is fundamental to deciphering function in basic and translational research. The chemical diversity of proteins is expressed in their many proteoforms, which result from combinations of genetic polymorphisms, RNA splice variants, and posttranslational modifications. This knowledge is foundational for the biological complexes and networks that control biology yet remains largely unknown. We propose here an ambitious initiative to define the human proteome, that is, to generate a definitive reference set of the proteoforms produced from the genome. Several examples of the power and importance of proteoform-level knowledge in disease-based research are presented along with a call for improved technologies in a two-pronged strategy to the Human Proteoform Project.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mandel-Brehm2021,

title = {Elevated N-Linked Glycosylation of IgG V Regions in Myasthenia Gravis Disease Subtypes},

author = {Mandel-Brehm, C and Fichtner, M.L and Jiang, R and Winton, V.J and Vazquez, S.E and Pham, M.C and Hoehn, K.B and Kelleher, N.L and Nowak, R.J and Kleinstein, S.H and Wilson, M.R and DeRisi, J.L and O'Connor, K.C},

doi = {10.4049/jimmunol.2100225},

year  = {2021},

date = {2021-09-20},

journal = {J Immunol.},

volume = {207},

number = {8},

pages = {2005-2014},

abstract = {Elevated N-linked glycosylation of IgG V regions (IgG-VN-Glyc) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-VN-Glyc, we studied patients with distinct subtypes of myasthenia gravis (MG), a B cell-mediated autoimmune disease. Our experimental design focused on examining the B cell repertoire and total IgG. It specifically included adaptive immune receptor repertoire sequencing to quantify and characterize N-linked glycosylation sites in the circulating BCR repertoire, proteomics to examine glycosylation patterns of the total circulating IgG, and an exploration of human-derived recombinant autoantibodies, which were studied with mass spectrometry and Ag binding assays to respectively confirm occupation of glycosylation sites and determine whether they alter binding. We found that the frequency of IgG-VN-Glyc motifs was increased in the total BCR repertoire of patients with MG when compared with healthy donors. The elevated frequency was attributed to both biased V gene segment usage and somatic hypermutation. IgG-VN-Glyc could be observed in the total circulating IgG in a subset of patients with MG. Autoantigen binding, by four patient-derived MG autoantigen-specific mAbs with experimentally confirmed presence of IgG-VN-Glyc, was not altered by the glycosylation. Our findings extend prior work on patterns of Ig V region N-linked glycosylation in autoimmunity to MG subtypes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Park2021,

title = {Characterization of a Copper-Chelating Natural Product from the Methanotroph Methylosinus sp. LW3},

author = {Park, Y.J and Roberts, G.M and Montaser, R and Kenney, G.E and Thomas, P.M and Kelleher, N.L and Rosenzweig, A.C},

doi = {10.1021/acs.biochem.1c00443},

year  = {2021},

date = {2021-09-12},

journal = {Biochemistry.},

volume = {60},

number = {38},

pages = {2845-2850},

abstract = {Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptidic natural products that bind copper with high affinity. Methanotrophic bacteria use Mbns to acquire copper needed for enzymatic methane oxidation. Despite the presence of Mbn operons in a range of methanotroph and other bacterial genomes, few Mbns have been isolated and structurally characterized. Here we report the isolation of a novel Mbn from the methanotroph Methylosinus (Ms.) sp. LW3. Mass spectrometric and nuclear magnetic resonance spectroscopic data indicate that this Mbn, the largest characterized to date, consists of a 13-amino acid backbone modified to include pyrazinedione/oxazolone rings and neighboring thioamide groups derived from cysteine residues. The pyrazinedione ring is more stable to acid hydrolysis than the oxazolone ring and likely protects the Mbn from degradation. The structure corresponds exactly to that predicted on the basis of the Ms. sp. LW3 Mbn operon content, providing support for the proposed role of an uncharacterized biosynthetic enzyme, MbnF, and expanding the diversity of known Mbns.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Aoi2021,

title = {SPT5 stabilization of promoter-proximal RNA polymerase II},

author = {Aoi, Y and Takahashi, Y.H and Shah, A.P and Iwanaszko, M and Rendleman, E.J and Khan, N.H and Cho, B.K and Goo, Y.A and Ganesan, S and Kelleher, N.L and Shilatifard, A},

doi = {10.1016/j.molcel.2021.08.006},

year  = {2021},

date = {2021-09-03},

journal = {Mol Cell.},

volume = {81},

number = {21},

pages = {4413-4424.e5},

abstract = {Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Wilkins2021,

title = {Spectrum of Apolipoprotein AI and Apolipoprotein AII Proteoforms and Their Associations With Indices of Cardiometabolic Health: The CARDIA Study},

author = {Wilkins, J.T and Seckler, H.S and Rink, J and Compton, P.D and Fornelli, L and Thaxton, C.S and LeDuc, R and Jacobs, D and Doubleday, P.F and Sniderman, A and Lloyd-Jones, D.M and Kelleher, N.L},

doi = {10.1161/JAHA.120.019890},

year  = {2021},

date = {2021-09-02},

journal = {J Am Heart Assoc.},

volume = {10},

number = {17},

abstract = {Background ApoAI (apolipoproteins AI) and apoAII (apolipoprotein AII) are structural and functional proteins of high-density lipoproteins (HDL) which undergo post-translational modifications at specific residues, creating distinct proteoforms. While specific post-translational modifications have been reported to alter apolipoprotein function, the full spectrum of apoAI and apoAII proteoforms and their associations with cardiometabolic phenotype remains unknown. Herein, we comprehensively characterize apoAI and apoAII proteoforms detectable in serum and their post-translational modifications and quantify their associations with cardiometabolic health indices. Methods and Results Using top-down proteomics (mass-spectrometric analysis of intact proteins), we analyzed paired serum samples from 150 CARDIA (Coronary Artery Risk Development in Young Adults) study participants from year 20 and 25 exams. Measuring 15 apoAI and 9 apoAII proteoforms, 6 of which carried novel post-translational modifications, we quantified associations between percent proteoform abundance and key cardiometabolic indices. Canonical (unmodified) apoAI had inverse associations with HDL cholesterol and HDL-cholesterol efflux, and positive associations with obesity indices (body mass index, waist circumference), and triglycerides, whereas glycated apoAI showed positive associations with serum glucose and diabetes mellitus. Fatty-acid‒modified ApoAI proteoforms had positive associations with HDL cholesterol and efflux, and inverse associations with obesity indices and triglycerides. Truncated and dimerized proteoforms of apoAII were associated with HDL cholesterol (positively) and obesity indices (inversely). Several proteoforms had no significant associations with phenotype. Conclusions Associations between apoAI and AII and cardiometabolic indices are proteoform-specific. These results provide "proof-of-concept" that precise chemical characterization of human apolipoproteins will yield improved insights into the complex pathways through which proteins signify and mediate health and disease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Doubleday2021,

title = {Oncogenic KRAS creates an aspartate metabolism signature in colorectal cancer cells},

author = {Doubleday, P.F and Fornelli, L and Ntai, I and Kelleher, N.L},

doi = {10.1111/febs.16111},

year  = {2021},

date = {2021-07-27},

journal = {FEBS J.},

volume = {288},

number = {23},

pages = {6683-6699},

abstract = {Oncogenic mutations in the KRAS gene are found in 30-50% of colorectal cancers (CRC), and recent findings have demonstrated independent and nonredundant roles for wild-type and mutant KRAS alleles in governing signaling and metabolism. Here, we quantify proteomic changes manifested by KRAS mutation and KRAS allele loss in isogenic cell lines. We show that the expression of KRASG13D upregulates aspartate metabolizing proteins including PCK1, PCK2, ASNS, and ASS1. Furthermore, differential expression analyses of transcript-level data from CRC tumors identified the upregulation of urea cycle enzymes in CRC. We find that expression of ASS1 supports colorectal cancer cell proliferation and promotes tumor formation in vitro. We show that loss of ASS1 can be rescued with high levels of several metabolites.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Melani2021,

title = {Next-generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire},

author = {Melani, R.D and Des Soye, B.J and Kafader, J.O and Forte, E and Hollas, M and Blagojevic, V and Negrão, F and McGee, J.P and Drown, B and Lloyd-Jones, C and Seckler, H.S and Camarillo, J.M and Compton, P.D and LeDuc, R.D and Early, B and Fellers, R.T and Cho, B.K and Mattamana, B.B and Goo, Y.A and Thomas, P.M and Ash, M.K and Bhimalli, P.P and Al-Harthi, L and Sha, B.E and Schneider, J.R and Kelleher, N.L},

doi = {10.1021/acs.jproteome.1c00882},

year  = {2021},

date = {2021-07-07},

journal = {medRxiv.},

volume = {21},

number = {1},

pages = {274-288},

abstract = {Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS , a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multi-parametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We apply Ig-MS to plasma from subjects with severe & mild COVID-19, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, with compatibility to any recombinant antigen to gauge our immune responses to vaccination, pathogens, or autoimmune disorders.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Seckler2021,

title = {New Interface for Faster Proteoform Analysis: Immunoprecipitation Coupled with SampleStream-Mass Spectrometry},

author = {Santos Seckler, H.D and Park, H.M and Lloyd-Jones, C.M and Melani, R.D and Camarillo, J.M and Wilkins, J.T and Compton, P.D and Kelleher, N.L},

doi = {10.1021/jasms.1c00026},

year  = {2021},

date = {2021-07-07},

journal = {J Am Soc Mass Spectrom.},

volume = {32},

number = {7},

pages = {1659-1670},

abstract = {Different proteoform products of the same gene can exhibit differing associations with health and disease, and their patterns of modifications may offer more precise markers of phenotypic differences between individuals. However, currently employed protein-biomarker discovery and quantification tools, such as bottom-up proteomics and ELISAs, are mostly proteoform-unaware. Moreover, the current throughput for proteoform-level analyses by liquid chromatography mass spectrometry (LCMS) for quantitative top-down proteomics is incompatible with population-level biomarker surveys requiring robust, faster proteoform analysis. To this end, we developed immunoprecipitation coupled to SampleStream mass spectrometry (IP-SampleStream-MS) as a high-throughput, automated technique for the targeted quantification of proteoforms. We applied IP-SampleStream-MS to serum samples of 25 individuals to assess the proteoform abundances of apolipoproteins A-I (ApoA-I) and C-III (ApoC-III). The results for ApoA-I were compared to those of LCMS for these individuals, with IP-SampleStream-MS showing a >7-fold higher throughput with >50% better analytical variation. Proteoform abundances measured by IP-SampleStream-MS correlated strongly to LCMS-based values (R2 = 0.6-0.9) and produced convergent proteoform-to-phenotype associations, namely, the abundance of canonical ApoA-I was associated with lower HDL-C (R = 0.5) and glycated ApoA-I with higher fasting glucose (R = 0.6). We also observed proteoform-to-phenotype associations for ApoC-III, 22 glycoproteoforms of which were characterized in this study. The abundance of ApoC-III modified by a single N-acetyl hexosamine (HexNAc) was associated with indices of obesity, such as BMI, weight, and waist circumference (R ∼ 0.7). These data show IP-SampleStream-MS to be a robust, scalable workflow for high-throughput associations of proteoforms to phenotypes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Shen2021,

title = {Turnover and Inactivation Mechanisms for ( S)-3-Amino-4,4-difluorocyclopent-1-enecarboxylic Acid, a Selective Mechanism-Based Inactivator of Human Ornithine Aminotransferase},

author = {Shen, S and Butrin, A and Doubleday, P.F and Melani, R.D and Beaupre, B.A and Tavares, M.T and Ferreira, G.M and Kelleher, N.L and Moran, G.R and Liu, D and Silverman, R.B},

doi = {10.1021/jacs.1c02456},

year  = {2021},

date = {2021-06-16},

journal = {J Am Chem Soc.},

volume = {143},

number = {23},

pages = {8689-8703},

abstract = {The inhibition of human ornithine δ-aminotransferase (hOAT) is a potential therapeutic approach to treat hepatocellular carcinoma. In this work, (S)-3-amino-4,4-difluorocyclopent-1-enecarboxylic acid (SS-1-148, 6) was identified as a potent mechanism-based inactivator of hOAT while showing excellent selectivity over other related aminotransferases (e.g., GABA-AT). An integrated mechanistic study was performed to investigate the turnover and inactivation mechanisms of 6. A monofluorinated ketone (M10) was identified as the primary metabolite of 6 in hOAT. By soaking hOAT holoenzyme crystals with 6, a precursor to M10 was successfully captured. This gem-diamine intermediate, covalently bound to Lys292, observed for the first time in hOAT/ligand crystals, validates the turnover mechanism proposed for 6. Co-crystallization yielded hOAT in complex with 6 and revealed a novel noncovalent inactivation mechanism in hOAT. Native protein mass spectrometry was utilized for the first time in a study of an aminotransferase inactivator to validate the noncovalent interactions between the ligand and the enzyme; a covalently bonded complex was also identified as a minor form observed in the denaturing intact protein mass spectrum. Spectral and stopped-flow kinetic experiments supported a lysine-assisted E2 fluoride ion elimination, which has never been observed experimentally in other studies of related aminotransferase inactivators. This elimination generated the second external aldimine directly from the initial external aldimine, rather than the typical E1cB elimination mechanism, forming a quinonoid transient state between the two external aldimines. The use of native protein mass spectrometry, X-ray crystallography employing both soaking and co-crystallization methods, and stopped-flow kinetics allowed for the detailed elucidation of unusual turnover and inactivation pathways.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhu202,

title = {Remarkable and Unexpected Mechanism for ( S)-3-Amino-4-(difluoromethylenyl)cyclohex-1-ene-1-carboxylic Acid as a Selective Inactivator of Human Ornithine Aminotransferase},

author = {Zhu, W and Doubleday, P.F and Butrin, A and Weerawarna, P.M and Melani, R.D and Catlin, D.S and Dwight, T.A and Liu, D and Kelleher, N.L and Silverman, R.B},

doi = {10.1021/jacs.1c03572},

year  = {2021},

date = {2021-06-02},

journal = {J Am Chem Soc.},

volume = {143},

number = {21},

pages = {8193-8207},

abstract = {Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that was recently found to play an important role in the metabolic reprogramming of hepatocellular carcinoma (HCC) via the proline and glutamine metabolic pathways. The selective inhibition of hOAT by compound 10 exhibited potent in vivo antitumor activity. Inspired by the discovery of the aminotransferase inactivator (1S,3S)-3-amino-4-(difluoromethylene)cyclopentane-1-carboxylic acid (5), we rationally designed, synthesized, and evaluated a series of six-membered-ring analogs. Among them, 14 was identified as a new selective hOAT inactivator, which demonstrated a potency 22× greater than that of 10. Three different types of protein mass spectrometry approaches and two crystallographic approaches were employed to identify the structure of hOAT-14 and the formation of a remarkable final adduct (32') in the active site. These spectral studies reveal an enzyme complex heretofore not observed in a PLP-dependent enzyme, which has covalent bonds to two nearby residues. Crystal soaking experiments and molecular dynamics simulations were carried out to identify the structure of the active-site intermediate 27' and elucidate the order of the two covalent bonds that formed, leading to 32'. The initial covalent reaction of the activated warhead occurs with *Thr322 from the second subunit, followed by a subsequent nucleophilic attack by the catalytic residue Lys292. The turnover mechanism of 14 by hOAT was supported by a mass spectrometric analysis of metabolites and fluoride ion release experiments. This novel mechanism for hOAT with 14 will contribute to the further rational design of selective inactivators and an understanding of potential inactivation mechanisms by aminotransferases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Alfaro2021,

title = {The emerging landscape of single-molecule protein sequencing technologies},

author = {Alfaro, J.A and Bohländer, P and Dai, M and Filius, M and Howard, C.J and van Kooten, X.F and Ohayon, S and Pomorski, A and Schmid, S and Aksimentiev, A and Anslyn, E.V and Bedran, G and Cao, C and Chinappi, M and Coyaud, E and Dekker, C and Dittmar, G and Drachman, N and Eelkema, R and Goodlett, D and Hentz, S and Kalathiya, U and Kelleher, N.L and Kelly, R.T and Kelman, Z and Kim, S.H and Kuster, B and Rodriguez-Larrea, D and Lindsay, S and Maglia, G and Marcotte, E.M and Marino, J.P and Masselon, C and Mayer, M and Samaras, P and Sarthak, K and Sepiashvili, L and Stein, D and Wanunu, M and Wilhelm, M and Yin, P and Meller, A and Joo, C},

url = {https://pubmed.ncbi.nlm.nih.gov/34099939/},

doi = {10.1038/s41592-021-01143-1},

year  = {2021},

date = {2021-06-01},

urldate = {2021-06-01},

journal = {Nat Methods.},

volume = {18},

number = {6},

pages = {604-617},

abstract = {Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Adams2021,

title = {Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry},

author = {Adams, L.M and DeHart, C.J and Kelleher, N.L},

doi = {10.1007/978-1-0716-1190-6_3},

year  = {2021},

date = {2021-06-01},

journal = {Methods Mol Biol.},

number = {2262},

pages = {47-64},

abstract = {The characterization of biologically relevant post-translational modifications (PTMs) on KRAS4B has historically been carried out through methodologies such as immunoblotting with PTM-specific antibodies or peptide-based proteomic methods. While these methods have the potential to identify a given PTM on KRAS4B, they are incapable of characterizing or distinguishing the different molecular forms or proteoforms of KRAS4B from those of related RAS isoforms. We present a method that combines immunoprecipitation of KRAS4B with top-down mass spectrometry (IP-TDMS), thus enabling the precise characterization of intact KRAS4B proteoforms. We provide detailed protocols for the IP, LC-MS/MS, and data analysis comprising a successful IP-TDMS assay in the contexts of cancer cell lines and tissue samples.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Robey2021,

title = {An interpreted atlas of biosynthetic gene clusters from 1,000 fungal genomes},

author = {Robey, M.T and Caesar, L.K and Drott, M.T and Keller, N.P and Kelleher, N.L},

doi = {10.1073/pnas.2020230118},

year  = {2021},

date = {2021-05-11},

journal = {Proc Natl Acad Sci U S A.},

volume = {118},

number = {19},

abstract = {Fungi are prolific producers of natural products, compounds which have had a large societal impact as pharmaceuticals, mycotoxins, and agrochemicals. Despite the availability of over 1,000 fungal genomes and several decades of compound discovery efforts from fungi, the biosynthetic gene clusters (BGCs) encoded by these genomes and the associated chemical space have yet to be analyzed systematically. Here, we provide detailed annotation and analyses of fungal biosynthetic and chemical space to enable genome mining and discovery of fungal natural products. Using 1,037 genomes from species across the fungal kingdom (e.g., Ascomycota, Basidiomycota, and non-Dikarya taxa), 36,399 predicted BGCs were organized into a network of 12,067 gene cluster families (GCFs). Anchoring these GCFs with reference BGCs enabled automated annotation of 2,026 BGCs with predicted metabolite scaffolds. We performed parallel analyses of the chemical repertoire of fungi, organizing 15,213 fungal compounds into 2,945 molecular families (MFs). The taxonomic landscape of fungal GCFs is largely species specific, though select families such as the equisetin GCF are present across vast phylogenetic distances with parallel diversifications in the GCF and MF. We compare these fungal datasets with a set of 5,453 bacterial genomes and their BGCs and 9,382 bacterial compounds, revealing dramatic differences between bacterial and fungal biosynthetic logic and chemical space. These genomics and cheminformatics analyses reveal the large extent to which fungal and bacterial sources represent distinct compound reservoirs. With a >10-fold increase in the number of interpreted strains and annotated BGCs, this work better regularizes the biosynthetic potential of fungi for rational compound discovery.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Schachner2021,

title = {Reassembling protein complexes after controlled disassembly by top-down mass spectrometry in native mode},

author = {Schachner, L.F and Tran, D.P and Lee, A and McGee, J.P and Jooss, K and Durbin, K and Seckler, H.D.S and Adams, L and Cline, E and Melani, R and Ives, A.N and Des Soye, B and Kelleher, N.L and Patrie, S.M},

doi = {10.1016/j.ijms.2021.116591},

year  = {2021},

date = {2021-03-27},

journal = {Int J Mass Spectrom.},

volume = {465},

abstract = {The combined use of electrospray ionization run in so-called "native mode" with top-down mass spectrometry (nTDMS) is enhancing both structural biology and discovery proteomics by providing three levels of information in a single experiment: the intact mass of a protein or complex, the masses of its subunits and non-covalent cofactors, and fragment ion masses from direct dissociation of subunits that capture the primary sequence and combinations of diverse post-translational modifications (PTMs). While intact mass data are readily deconvoluted using well-known software options, the analysis of fragmentation data that result from a tandem MS experiment - essential for proteoform characterization - is not yet standardized. In this tutorial, we offer a decision-tree for the analysis of nTDMS experiments on protein complexes and diverse bioassemblies. We include an overview of strategies to navigate this type of analysis, provide example data sets, and highlight software for the hypothesis-driven interrogation of fragment ions for localization of PTMs, metals, and cofactors on native proteoforms. Throughout we have emphasized the key features (deconvolution, search mode, validation, other) that the reader can consider when deciding upon their specific experimental and data processing design using both open-access and commercial software.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{McGee2021,

title = {Isotopic Resolution of Protein Complexes up to 466 kDa Using Individual Ion Mass Spectrometry},

author = {McGee, J.P and Melani, R.D and Yip, P.F and Senko, M.W and Compton, P.D and Kafader, J.O and Kelleher, N.L},

url = {https://pubs.acs.org/doi/10.1021/acs.analchem.0c03282},

doi = {10.1021/acs.analchem.0c03282},

year  = {2021},

date = {2021-02-09},

urldate = {2021-02-09},

journal = {Anal Chem.},

volume = {93},

number = {5},

pages = {2723-2727},

abstract = {Native mass spectrometry involves transferring large biomolecular complexes into the gas phase, enabling the characterization of their composition and stoichiometry. However, the overlap in distributions created by residual solvation, ionic adducts, and post-translational modifications creates a high degree of complexity that typically goes unresolved at masses above ∼150 kDa. Therefore, native mass spectrometry would greatly benefit from higher resolution approaches for intact proteins and their complexes. By recording mass spectra of individual ions via charge detection mass spectrometry, we report isotopic resolution for pyruvate kinase (232 kDa) and β-galactosidase (466 kDa), extending the limits of isotopic resolution for high mass and high m/z by >2.5-fold and >1.6-fold, respectively.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Liu2021,

title = {In vitro-Constructed Ribosomes Enable Multi-site Incorporation of Noncanonical Amino Acids into Proteins},

author = {Liu, Y and Davis, R.G and Thomas, P.M and Kelleher, N.L and Jewett, M.C},

url = {https://pubs.acs.org/doi/10.1021/acs.biochem.0c00829},

doi = {10.1021/acs.biochem.0c00829},

year  = {2021},

date = {2021-01-26},

journal = {Biochemistry.},

volume = {60},

number = {3},

pages = {161-169},

abstract = {Efforts to expand the scope of ribosome-mediated polymerization to incorporate noncanonical amino acids (ncAAs) into peptides and proteins hold promise for creating new classes of enzymes, therapeutics, and materials. Recently, the integrated synthesis, assembly, and translation (iSAT) system was established to construct functional ribosomes in cell-free systems. However, the iSAT system has not been shown to be compatible with genetic code expansion. Here, to address this gap, we develop an iSAT platform capable of manufacturing pure proteins with site-specifically incorporated ncAAs. We first establish an iSAT platform based on extracts from genomically recoded Escherichia coli lacking release factor 1 (RF-1). This permits complete reassignment of the amber codon translation function. Next, we optimize orthogonal translation system components to demonstrate the benefits of genomic RF-1 deletion on incorporation of ncAAs into proteins. Using our optimized platform, we demonstrate high-level, multi-site incorporation of p-acetyl-phenylalanine (pAcF) and p-azido-phenylalanine into superfolder green fluorescent protein (sfGFP). Mass spectrometry analysis confirms the high accuracy of incorporation for pAcF at one, two, and five amber sites in sfGFP. The iSAT system updated for ncAA incorporation sets the stage for investigating ribosomal mutations to better understand the fundamental basis of protein synthesis, manufacturing proteins with new properties, and engineering ribosomes for novel polymerization chemistries.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Yusufova2021,

title = {Histone H1 loss drives lymphoma by disrupting 3D chromatin architecture},

author = {Yusufova, N and Kloetgen, A and Teater, M and Osunsade, A and Camarillo, J.M and Chin, C.R and Doane, A.S and Venters, B.J and Portillo-Ledesma, S and Conway, J and Phillip, J.M and Elemento, O and Scott, D.W and Béguelin, W and Licht, J.D and Kelleher, N.L and Staudt, L.M and Skoultchi, A.I and Keogh, M.C and Apostolou, E and Mason, C.E and Imielinski, M and Schlick, T and David, Y and Tsirigos, A and Allis, C.D and Soshnev, A.A and Cesarman, E and Melnick, A.M},

url = {https://www.nature.com/articles/s41586-020-3017-y},

doi = {10.1038/s41586-020-3017-y},

year  = {2021},

date = {2021-01-01},

urldate = {2021-01-01},

journal = {Nature.},

volume = {589},

number = {7841},

pages = {299-305},

abstract = {Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction1, although their biological functions are poorly understood. Mutations in the genes that encode H1 isoforms B-E (H1B, H1C, H1D and H1E; also known as H1-5, H1-2, H1-3 and H1-4, respectively) are highly recurrent in B cell lymphomas, but the pathogenic relevance of these mutations to cancer and the mechanisms that are involved are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in a profound architectural remodelling of the genome, which is characterized by large-scale yet focal shifts of chromatin from a compacted to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily owing to a gain of histone H3 dimethylation at lysine 36 (H3K36me2) and/or loss of repressive H3 trimethylation at lysine 27 (H3K27me3). These changes unlock the expression of stem cell genes that are normally silenced during early development. In mice, loss of H1c and H1e (also known as H1f2 and H1f4, respectively) conferred germinal centre B cells with enhanced fitness and self-renewal properties, ultimately leading to aggressive lymphomas with an increased repopulating potential. Collectively, our data indicate that H1 proteins are normally required to sequester early developmental genes into architecturally inaccessible genomic compartments. We also establish H1 as a bona fide tumour suppressor and show that mutations in H1 drive malignant transformation primarily through three-dimensional genome reorganization, which leads to epigenetic reprogramming and derepression of developmentally silenced genes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hoofnagle2020,

title = {Should LC-MS/MS Be the Reference Measurement Procedure to Determine Protein Concentrations in Human Samples?},

author = {Hoofnagle, A.N and Cobbaert, C.M and Delatour, V and Kelleher, N.L and Lowenthal, M.S and Shuford, C.M},

url = {https://academic.oup.com/clinchem/advance-article/doi/10.1093/clinchem/hvaa256/6024904},

doi = {10.1093/clinchem/hvaa256},

year  = {2020},

date = {2020-12-03},

journal = {Clin Chem.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hamm2020,

title = {Streptomyces buecherae sp. nov., an actinomycete isolated from multiple bat species},

author = {Hamm, P.S and Dunlap, C.A and Mullowney, M.W and Caimi, N.A and Kelleher, N.L and Thomson, R.J and Porras-Alfaro, A and Northup, D.E},

url = {https://link.springer.com/article/10.1007/s10482-020-01493-4},

doi = {10.1007/s10482-020-01493-4},

year  = {2020},

date = {2020-12-01},

journal = {Antonie Van Leeuwenhoek.},

volume = {113},

number = {12},

pages = {2213-2221},

abstract = {A putative novel clade within the genus Streptomyces was discovered following antifungal screening against Pseudogymnoascus destructans, the causative agent of white-nose syndrome, and described using multi-locus sequencing analysis. Swabs from both the cave myotis bat (Myotis velifer) and the Brazilian free-tailed bat (Tadarida brasiliensis) in southern New Mexico bore isolates AC536, AC541T and AC563, which were characterised using phylogenetic, morphological, and phenotypic analyses. Multi-locus sequence analysis positions AC541T with neighbors Streptomyces rubidus (NRRL B-24619T), Streptomyces guanduensis (NRRL B-24617T), and Streptomyces yeochonensis (NRRL B-24245T). A complete genome of the type strain was assembled to determine its taxonomy and secondary metabolite potential. ANI comparisons between all closely related types strains are shown to be well below the 95-96% species delineation. DNA-DNA relatedness between AC541T and its nearest neighbors ranged between 23.7 and 24.1% confirming novelty. Approximately 1.49 Mb or 17.76% of the whole genome is devoted to natural product biosynthesis. The DNA G + C content of the genomic DNA of the type strain is 73.13 mol %. Micromorphology depicts ovoid spores with smooth surfaces in flexuous chains. Strains presented an ivory to yellow hue on most ISP media except inorganic salts-starch agar (ISP4) and can grow on D-glucose, mannitol, and D-fructose, but exhibited little to no growth on L-arabinose, sucrose, D-xylose, inositol, L-rhamnose, D-raffinose, and cellulose. This clade possesses the capability to grow from 10 to 45 °C and 12.5% (w/v) NaCl. There was strain growth variation in pH, but all isolates thrive at alkaline levels. Based on our polyphasic study of AC541T, the strain warrants the assignment to a novel species, for which the name Streptomyces buecherae sp. nov. is proposed. The type strain is AC541T (= JCM 34263T, = ATCC TSD201T).},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{An2020b,

title = {Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma},

author = {An, S and Camarillo, J.M and Huang, T.Y and Li, D and Morris, J.A and Zoltek, M.A and Qi, J and Behbahani, M and Kambhampati, M and Kelleher, N.L and Nazarian, J and Thomas, P.M and Saratsis, A.M},

url = {https://jeccr.biomedcentral.com/articles/10.1186/s13046-020-01773-x},

doi = {10.1186/s13046-020-01773-x},

year  = {2020},

date = {2020-11-25},

journal = {J Exp Clin Cancer Res.},

volume = {39},

number = {1},

pages = {261},

abstract = {Background: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles.



Methods: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7).



Results: We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro.



Conclusion: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.



Keywords: DIPG; H3K27M; H3K36me2; H4K16ac; Histone.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Tryon2020,

title = {Genome Mining and Metabolomics Uncover a Rare d-Capreomycidine Containing Natural Product and Its Biosynthetic Gene Cluster},

author = {Tryon, J.H and Rote, J.C and Chen, L and Robey, M.T and Vega, M.M and Phua, W.C and Metcalf, W.W and Ju, K.S and Kelleher, N.L and Thomson, R.J},

url = {https://pubs.acs.org/doi/10.1021/acschembio.0c00663},

doi = {10.1021/acschembio.0c00663},

year  = {2020},

date = {2020-11-20},

journal = {ACS Chem Biol. },

volume = {15},

number = {11},

pages = {3013-3020},

abstract = {We report the metabolomics-driven genome mining of a new cyclic-guanidino incorporating non-ribosomal peptide synthetase (NRPS) gene cluster and full structure elucidation of its associated hexapeptide product, faulknamycin. Structural studies unveiled that this natural product contained the previously unknown (R,S)-stereoisomer of capreomycidine, d-capreomycidine. Furthermore, heterologous expression of the identified gene cluster successfully reproduces faulknamycin production without an observed homologue of VioD, the pyridoxal phosphate (PLP)-dependent enzyme found in all previous l-capreomycidine biosynthesis. An alternative NRPS-dependent pathway for d-capreomycidine biosynthesis is proposed.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Caesar2020b,

title = {In the fungus where it happens: History and future propelling Aspergillus nidulans as the archetype of natural products research},

author = {Caesar, L.K and Kelleher, N.L and Keller, N.P. },

url = {https://www.sciencedirect.com/science/article/pii/S1087184520301687?via%3Dihub},

doi = {10.1016/j.fgb.2020.103477},

year  = {2020},

date = {2020-11-01},

journal = {Fungal Genet Biol.},

volume = {144},

abstract = {In 1990 the first fungal secondary metabolite biosynthetic gene was cloned in Aspergillus nidulans. Thirty years later, >30 biosynthetic gene clusters (BGCs) have been linked to specific natural products in this one fungal species. While impressive, over half of the BGCs in A. nidulans remain uncharacterized and their compounds structurally and functionally unknown. Here, we provide a comprehensive review of past advances that have enabled A. nidulans to rise to its current status as a natural product powerhouse focusing on the discovery and annotation of secondary metabolite clusters. From genome sequencing, heterologous expression, and metabolomics to CRISPR and epigenetic manipulations, we present a guided tour through the evolution of technologies developed and utilized in the last 30 years. These insights provide perspective to future efforts to fully unlock the biosynthetic potential of A. nidulans and, by extension, the potential of other filamentous fungi.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Abshiru2020,

title = {Targeted detection and quantitation of histone modifications from 1,000 cells},

author = {Abshiru, N.A and Sikora, J.W and Camarillo, J.M and Morris, J.A and Compton, P.D and Lee, T and Neelamraju, Y and Haddox, S and Sheridan, C and Carroll, M and Cripe, L.D and Tallman, M.S and Paietta, E.M and Melnick, A.M and Thomas, P.M and Garrett-Bakelman, F.E and Kelleher, N.L. },

url = {https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240829},

doi = {10.1371/journal.pone.0240829},

year  = {2020},

date = {2020-10-26},

journal = {PLoS One.},

volume = {15},

number = {10},

abstract = {Histone post-translational modifications (PTMs) create a powerful regulatory mechanism for maintaining chromosomal integrity in cells. Histone acetylation and methylation, the most widely studied histone PTMs, act in concert with chromatin-associated proteins to control access to genetic information during transcription. Alterations in cellular histone PTMs have been linked to disease states and have crucial biomarker and therapeutic potential. Traditional bottom-up mass spectrometry of histones requires large numbers of cells, typically one million or more. However, for some cell subtype-specific studies, it is difficult or impossible to obtain such large numbers of cells and quantification of rare histone PTMs is often unachievable. An established targeted LC-MS/MS method was used to quantify the abundance of histone PTMs from cell lines and primary human specimens. Sample preparation was modified by omitting nuclear isolation and reducing the rounds of histone derivatization to improve detection of histone peptides down to 1,000 cells. In the current study, we developed and validated a quantitative LC-MS/MS approach tailored for a targeted histone assay of 75 histone peptides with as few as 10,000 cells. Furthermore, we were able to detect and quantify 61 histone peptides from just 1,000 primary human stem cells. Detection of 37 histone peptides was possible from 1,000 acute myeloid leukemia patient cells. We anticipate that this revised method can be used in many applications where achieving large cell numbers is challenging, including rare human cell populations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Deighan2020,

title = {Development of novel methods for non-canonical myeloma protein analysis with an innovative adaptation of immunofixation electrophoresis, native top-down mass spectrometry, and middle-down de novo sequencing},

author = {Deighan, W.I and Winton, V.J and Melani, R.D and Anderson, L.C and McGee, J.P and Schachner, L.F and Barnidge, D and Murray, D and Alexander, H.D and Gibson, D.S and Deery, M.J and McNicholl, F.P and McLaughlin, J and Kelleher, N.L and Thomas, P.M},

url = {https://www.degruyter.com/document/doi/10.1515/cclm-2020-1072/html},

doi = {10.1515/cclm-2020-1072},

year  = {2020},

date = {2020-10-20},

journal = {Clin Chem Lab Med.},

abstract = {Objectives: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins.



Methods: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator.



Results: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin.



Conclusions: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities.



Keywords: immunofixation electrophoresis; multiple myeloma; native mass spectrometry; top-down protein sequencing; truncated heavy chains.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Srzentić2020,

title = {Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.},

author = {Srzentić, K and Fornelli, L and Tsybin, Y.O and Loo, J.A and Seckler, H and Agar, J.N and Anderson, L.C and Bai, D.L and Beck, A and Brodbelt, J.S and van der Burgt, Y.E.M and Chamot-Rooke, J and Chatterjee, S and Chen, Y and Clarke, D.J and Danis, P.O and Diedrich, J.K and D'Ippolito, R.A and Dupré, M and Gasilova, N and Ge, Y and Goo, Y.A and Goodlett, D.R and Greer, S and Haselmann, K.F and He, L and Hendrickson, C.L and Hinkle, J.D and Holt, M.V and Hughes, S and Hunt, D.F and Kelleher, N.L and Kozhinov, A.N and Lin, Z and Malosse, C and Marshall, A.G and Menin, L and Millikin, R.J and Nagornov, K.O and Nicolardi, S and Paša-Tolić, L and Pengelley, S and Quebbemann, N.R and Resemann, A and Sandoval, W and Sarin, R and Schmitt, N.D and Shabanowitz, J and Shaw, J.B and Shortreed, M.R and Smith, L.M and Sobott, F and Suckau, D and Toby, T and Weisbrod, C.R and Wildburger, N.C and Yates, J.R 3rd and Yoon, S.H and Young, N.L and Zhou, M},

doi = {10.1021/jasms.0c00036},

year  = {2020},

date = {2020-09-02},

journal = {J Am Soc Mass Spectrom.},

volume = {31},

number = {9},

pages = {1783-1802},

abstract = {The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Caesar2020,

title = {Heterologous Expression of the Unusual Terreazepine Biosynthetic Gene Cluster Reveals a Promising Approach for Identifying New Chemical Scaffolds.},

author = {Caesar, L.K and Robey, M.T and Swyer, M and Islam, M.N and Ye, R and Vagadia, P.P and Schiltz, G.E and Thomas, P.M and Wu, C.C and Kelleher, N.L and Keller, N.P and Bok, J.W},

doi = {10.1128/mBio.01691-20.},

year  = {2020},

date = {2020-08-25},

journal = {mBio.},

volume = {11},

number = {4},

abstract = {Advances in genome sequencing have revitalized natural product discovery efforts, revealing the untapped biosynthetic potential of fungi. While the volume of genomic data continues to expand, discovery efforts are slowed due to the time-consuming nature of experiments required to characterize new molecules. To direct efforts toward uncharacterized biosynthetic gene clusters most likely to encode novel chemical scaffolds, we took advantage of comparative metabolomics and heterologous gene expression using fungal artificial chromosomes (FACs). By linking mass spectral profiles with structural clues provided by FAC-encoded gene clusters, we targeted a compound originating from an unusual gene cluster containing an indoleamine 2,3-dioxygenase (IDO). With this approach, we isolate and characterize R and S forms of the new molecule terreazepine, which contains a novel chemical scaffold resulting from cyclization of the IDO-supplied kynurenine. The discovery of terreazepine illustrates that FAC-based approaches targeting unusual biosynthetic machinery provide a promising avenue forward for targeted discovery of novel scaffolds and their biosynthetic enzymes, and it also represents another example of a biosynthetic gene cluster "repurposing" a primary metabolic enzyme to diversify its secondary metabolite arsenal.IMPORTANCE Here, we provide evidence that Aspergillus terreus encodes a biosynthetic gene cluster containing a repurposed indoleamine 2,3-dioxygenase (IDO) dedicated to secondary metabolite synthesis. The discovery of this neofunctionalized IDO not only enabled discovery of a new compound with an unusual chemical scaffold but also provided insight into the numerous strategies fungi employ for diversifying and protecting themselves against secondary metabolites. The observations in this study set the stage for further in-depth studies into the function of duplicated IDOs present in fungal biosynthetic gene clusters and presents a strategy for accessing the biosynthetic potential of gene clusters containing duplicated primary metabolic genes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ives2020,

title = {Using 10,000 Fragment Ions to Inform Scoring in Native Top-down Proteomics.},

author = {Ives, A.N and Su, T and Durbin, K.R and Early, B.P and Dos Santos Seckler, H and Fellers, R.T and LeDuc, R.D and Schachner, L.F and Patrie, S.M and Kelleher, N.L},

doi = {10.1021/jasms.0c00026},

year  = {2020},

date = {2020-07-01},

journal = {J Am Soc Mass Spectrom.},

volume = {31},

number = {7},

pages = {1398-1409},

abstract = {Protein fragmentation is a critical component of top-down proteomics, enabling gene-specific protein identification and full proteoform characterization. The factors that influence protein fragmentation include precursor charge, structure, and primary sequence, which have been explored extensively for collision-induced dissociation (CID). Recently, noticeable differences in CID-based fragmentation were reported for native versus denatured proteins, motivating the need for scoring metrics that are tailored specifically to native top-down mass spectrometry (nTDMS). To this end, position and intensity were tracked for 10,252 fragment ions produced by higher-energy collisional dissociation (HCD) of 159 native monomers and 70 complexes. We used published structural data to explore the relationship between fragmentation and protein topology and revealed that fragmentation events occur at a large range of relative residue solvent accessibility. Additionally, our analysis found that fragment ions at sites with an N-terminal aspartic acid or a C-terminal proline make up on average 40 and 27%, respectively, of the total matched fragment ion intensity in nTDMS. Percent intensity contributed by each amino acid was determined and converted into weights to (1) update the previously published C-score and (2) construct a native Fragmentation Propensity Score. Both scoring systems showed an improvement in protein identification or characterization in comparison to traditional methods and overall increased confidence in results with fewer matched fragment ions but with high probability nTDMS fragmentation patterns. Given the rise of nTDMS as a tool for structural mass spectrometry, we forward these scoring metrics as new methods to enhance analysis of nTDMS data.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Béguelin2020,

title = {Mutant EZH2 Induces a Pre-malignant Lymphoma Niche by Reprogramming the Immune Response.},

author = {Béguelin, W and Teater, M and Meydan, C and Hoehn, K.B and Phillip, J.M and Soshnev, A.A and Venturutti, L and Rivas, M.A and Calvo-Fernández, M.T and Gutierrez, J and Camarillo, J.M and Takata, K and Tarte, K and Kelleher, N.L and Steidl, C and Mason, C.E and Elemento, O and Allis, C.D and Kleinstein, S.H and Melnick, A.M},

doi = {10.1016/j.ccell.2020.04.004.},

year  = {2020},

date = {2020-05-11},

journal = {Cancer Cell},

volume = {37},

number = {5},

pages = {655-673},

abstract = {Follicular lymphomas (FLs) are slow-growing, indolent tumors containing extensive follicular dendritic cell (FDC) networks and recurrent EZH2 gain-of-function mutations. Paradoxically, FLs originate from highly proliferative germinal center (GC) B cells with proliferation strictly dependent on interactions with T follicular helper cells. Herein, we show that EZH2 mutations initiate FL by attenuating GC B cell requirement for T cell help and driving slow expansion of GC centrocytes that become enmeshed with and dependent on FDCs. By impairing T cell help, mutant EZH2 prevents induction of proliferative MYC programs. Thus, EZH2 mutation fosters malignant transformation by epigenetically reprograming B cells to form an aberrant immunological niche that reflects characteristic features of human FLs, explaining how indolent tumors arise from GC B cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kafader2020c,

title = {Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes.},

author = {Kafader, J.O and Melani, R.D and Durbin, K.R and Ikwuagwu, B and Early, B.P and Fellers, R.T and Beu, S.C and Zabrouskov, V and Makarov, A.A and Maze, J.T and Shinholt, D.L and Yip, P.F and Tullman-Ercek, D and Senko, M.W and Compton, P.D and Kelleher, N.L},

doi = {10.1038/s41592-020-0764-5},

year  = {2020},

date = {2020-04-01},

journal = {Nat Methods.},

volume = {17},

number = {4},

pages = {391-394},

abstract = {An Orbitrap-based ion analysis procedure determines the direct charge for numerous individual protein ions to generate true mass spectra. This individual ion mass spectrometry (I2MS) method for charge detection enables the characterization of highly complicated mixtures of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by typical measurements of ensembles of ions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhu2020,

title = {A Remarkable Difference That One Fluorine Atom Confers on the Mechanisms of Inactivation of Human Ornithine Aminotransferase by Two Cyclohexene Analogues of γ-Aminobutyric Acid.},

author = {Zhu, W and Doubleday, P.F and Catlin, D.S and Weerawarna, P.M and Butrin, A and Shen, S and Wawrzak, Z and Kelleher, N.L and Liu, D and Silverman, R.B},

doi = {10.1021/jacs.0c00193},

year  = {2020},

date = {2020-03-11},

journal = {J Am Chem Soc.},

volume = {142},

number = {10},

pages = {4892-4903},

abstract = {Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate-dependent enzyme, plays a critical role in the progression of hepatocellular carcinoma (HCC). Pharmacological selective inhibition of hOAT has been shown to be a potential therapeutic approach for HCC. Inspired by the discovery of the nonselective aminotransferase inactivator (1R,3S,4S)-3-amino-4-fluoro cyclopentane-1-carboxylic acid (1), in this work, we rationally designed, synthesized, and evaluated a novel series of fluorine-substituted cyclohexene analogues, thereby identifying 8 and 9 as novel selective hOAT time-dependent inhibitors. Intact protein mass spectrometry and protein crystallography demonstrated 8 and 9 as covalent inhibitors of hOAT, which exhibit two distinct inactivation mechanisms resulting from the difference of a single fluorine atom. Interestingly, they share a similar turnover mechanism, according to the mass spectrometry-based analysis of metabolites and fluoride ion release experiments. Molecular dynamics (MD) simulations and electrostatic potential (ESP) charge calculations were conducted, which elucidated the significant influence of the one-fluorine difference on the corresponding intermediates, leading to two totally different inactivation pathways. The novel addition-aromatization inactivation mechanism for 9 contributes to its significantly enhanced potency, along with excellent selectivity over other aminotransferases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kafader2020b,

title = {Individual Ion Mass Spectrometry Enhances the Sensitivity and Sequence Coverage of Top-Down Mass Spectrometry.},

author = {Kafader, J.O and Durbin, K.R and Melani, R.D and Des Soye, B.J and Schachner, L.F and Senko, M.W and Compton, P.D and Kelleher, N.L},

doi = {10.1021/acs.jproteome.9b00797},

year  = {2020},

date = {2020-03-06},

journal = {J Proteome Res.},

volume = {19},

number = {3},

pages = {1346-1350},

abstract = {Charge detection mass spectrometry (CDMS) is mainly utilized to determine the mass of intact molecules. Previous applications of CDMS have determined the mass-to-charge ratio and the charge of large polymers, DNA molecules, and native protein complexes, from which corresponding mass values could be assigned. Recent advances have demonstrated that CDMS using an Orbitrap mass analyzer yields the reliable assignment of integer charge states that enables individual ion mass spectrometry (I2MS) and spectral output directly into the mass domain. Here I2MS analysis was extended to isotopically resolved fragment ions from intact proteoforms for the first time. With a radically different bias for ion readout, I2MS identified low-abundance fragment ions containing many hundreds of residues that were undetectable by standard Orbitrap measurements, leading to a doubling in the sequence coverage of triosephosphate isomerase. Thus MS/MS with the detection of individual ions (MS/I2MS) provides a far greater ability to detect high mass fragment ions and exhibits strong complementarity to traditional spectral readout in this, its first application to top-down mass spectrometry.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{McGee2020,

title = {Voltage Rollercoaster Filtering of Low-Mass Contaminants During Native Protein Analysis.},

author = {McGee, J.P and Melani, R.D and Goodwin, M and McAlister, G and Huguet, R and Senko, M.W and Compton, P.D and Kelleher, N.L},

doi = {10.1021/jasms.9b00037},

year  = {2020},

date = {2020-03-04},

journal = {J Am Soc Mass Spectrom.},

volume = {31},

number = {3},

pages = {763-767},

abstract = {Intact protein mass spectrometry (MS) via electrospray-based methods is often degraded by low-mass contaminants, which can suppress the spectral quality of the analyte of interest via space-charge effects. Consequently, selective removal of contaminants by their mobilities would benefit native MS if achieved without additional hardware and before the mass analyzer regions used for selection, analyte readout, or tandem MS. Here, we use the high-pressure multipole within the source of an Orbitrap Tribrid as the foundation for a coarse ion filter. Using this method, we show complete filtration of 2 mM polyethylene glycol (PEG-1000) during native MS of SILu mAb antibody present at a 200× lower concentration. We also show the generality of the process by rescuing 10 μM tetrameric pyruvate kinase from 2 mM PEG-1000, asserting this voltage rollercoaster filtering (VRF) method for use in native MS as an efficient alternative to conventional purification methods.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kosciuk2020,

title = {NMT1 and NMT2 are lysine myristoyltransferases regulating the ARF6 GTPase cycle.},

author = {Kosciuk, T and Price, I.R and Zhang, X and Zhu, C and Johnson, K.N and Zhang, S and Halaby, S.L and Komaniecki, G.P and Yang, M and DeHart, C.J and Thomas, P.M and Kelleher, N.L and Fromme, J.C and Lin, H},

doi = {10.1038/s41467-020-14893-x.},

year  = {2020},

date = {2020-02-26},

journal = {Nat Commun.},

volume = {11},

number = {1},

pages = {1067},

abstract = {Lysine fatty acylation in mammalian cells was discovered nearly three decades ago, yet the enzymes catalyzing it remain unknown. Unexpectedly, we find that human N-terminal glycine myristoyltransferases (NMT) 1 and 2 can efficiently myristoylate specific lysine residues. They modify ADP-ribosylation factor 6 (ARF6) on lysine 3 allowing it to remain on membranes during the GTPase cycle. We demonstrate that the NAD+-dependent deacylase SIRT2 removes the myristoyl group, and our evidence suggests that NMT prefers the GTP-bound while SIRT2 prefers the GDP-bound ARF6. This allows the lysine myrisotylation-demyristoylation cycle to couple to and promote the GTPase cycle of ARF6. Our study provides an explanation for the puzzling dissimilarity of ARF6 to other ARFs and suggests the existence of other substrates regulated by this previously unknown function of NMT. Furthermore, we identified a NMT/SIRT2-ARF6 regulatory axis, which may offer new ways to treat human diseases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Shen2020,

title = {Mechanism-Based Design of 3-Amino-4-Halocyclopentenecarboxylic Acids as Inactivators of GABA Aminotransferase},

author = {Shen, S and Doubleday, P.F and Weerawarna, P.M and Zhu, W and Kelleher, N.L and Silverman, R.B},

url = {https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00672},

doi = {10.1021/acsmedchemlett.9b00672},

year  = {2020},

date = {2020-02-18},

journal = {ACS Med Chem Lett.},

volume = {11},

number = {10},

pages = {1949-1955},

abstract = {Aminotransferases are pyridoxal 5'-phosphate-dependent enzymes that catalyze reversible transamination reactions between an amino acid and an α-keto acid, playing a critical role in cellular nitrogen metabolism. It is evident that γ-aminobutyric acid aminotransferase (GABA-AT), which balances the levels of inhibitory and excitatory neurotransmitters, has emerged as a promising therapeutic target for epilepsy and cocaine addiction based on mechanism-based inactivators (MBIs). In this work, we established an integrated approach using computational simulation, organic synthesis, biochemical evaluation, and mass spectrometry to facilitate our design and mechanistic studies of MBIs, which led to the identification of a new cyclopentene-based analogue (6a), 25-times more efficient as an inactivator of GABA-AT compared to the parent compound (1R,3S,4S)-3-amino-4-fluorocyclopentane carboxylic acid (FCP, 4).},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Schneider2020,

title = {A MUC16 IgG Binding Activity Selects for a Restricted Subset of IgG Enriched for Certain Simian Immunodeficiency Virus Epitope Specificities.},

author = {Schneider, J.R and Shen, X and Orlandi, C and Nyanhete, T and Sawant, S and Carias, A.M and Smith, A.D 4th and Kelleher, N.L and Veazey, R.S and Lewis, G.K and Tomaras, G.D and Hope, T.J},

doi = {10.1128/JVI.01246-19},

year  = {2020},

date = {2020-02-14},

journal = {J Virol.},

volume = {94},

number = {5},

abstract = {We have recently shown that MUC16, a component of the glycocalyx of some mucosal barriers, has elevated binding to the G0 glycoform of the Fc portion of IgG. Therefore, IgG from patients chronically infected with human immunodeficiency virus (HIV), who typically exhibit increased amounts of G0 glycoforms, showed increased MUC16 binding compared to uninfected controls. Using the rhesus macaque simian immunodeficiency virus SIVmac251 model, we can compare plasma antibodies before and after chronic infection. We find increased binding of IgG to MUC16 after chronic SIV infection. Antibodies isolated for tight association with MUC16 (MUC16-eluted antibodies) show reduced FcγR engagement and antibody-dependent cellular cytotoxicity (ADCC) activity. The glycosylation profile of these IgGs was consistent with a decrease in FcγR engagement and subsequent ADCC effector function, as they contain a decrease in afucosylated bisecting glycoforms that preferentially bind FcγRs. Testing of the SIV antigen specificity of IgG from SIV-infected macaques revealed that the MUC16-eluted antibodies were enriched for certain specific epitopes, including regions of gp41 and gp120. This enrichment of specific antigen responses for fucosylated bisecting glycoforms and the subsequent association with MUC16 suggests that the immune response has the potential to direct specific epitope responses to localize to the glycocalyx through interaction with this specific mucin.IMPORTANCE Understanding how antibodies are distributed in the mucosal environment is valuable for developing a vaccine to block HIV infection. Here, we study an IgG binding activity in MUC16, potentially representing a new IgG effector function that would concentrate certain antibodies within the glycocalyx to trap pathogens before they can reach the underlying columnar epithelial barriers. These studies reveal that rhesus macaque IgG responses during chronic SIV infection generate increased antibodies that bind MUC16, and interestingly, these MUC16-tethered antibodies are enriched for binding to certain antigens. Therefore, it may be possible to direct HIV vaccine-generated responses to associate with MUC16 and enhance the antibody's ability to mediate immune exclusion by trapping virions within the glycocalyx and preventing the virus from reaching immune target cells within the mucosa. This concept will ultimately have to be tested in the rhesus macaque model, which is shown here to have MUC16-targeted antigen responses.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Fornelli2020,

title = {Thorough Performance Evaluation of 213 nm Ultraviolet Photodissociation for Top-down Proteomics},

author = {Fornelli, L and Srzentić, K and Toby, T.K and Doubleday, P.F and Huguet, R and Mullen, C and Melani, R.D and Dos Santos Seckler, H and DeHart, C.J and Weisbrod, C.R and Durbin, K.R and Greer, J.B and Early, B.P and Fellers, R.T and Zabrouskov, V and Thomas, P.M and Compton, P.D and Kelleher, N.L},

url = {https://www.mcponline.org/content/19/2/405.long},

doi = {10.1074/mcp.TIR119.001638},

year  = {2020},

date = {2020-02-01},

journal = {Mol Cell Proteomics.},

volume = {19},

number = {2},

pages = {405-420},

abstract = {Top-down proteomics studies intact proteoform mixtures and offers important advantages over more common bottom-up proteomics technologies, as it avoids the protein inference problem. However, achieving complete molecular characterization of investigated proteoforms using existing technologies remains a fundamental challenge for top-down proteomics. Here, we benchmark the performance of ultraviolet photodissociation (UVPD) using 213 nm photons generated by a solid-state laser applied to the study of intact proteoforms from three organisms. Notably, the described UVPD setup applies multiple laser pulses to induce ion dissociation, and this feature can be used to optimize the fragmentation outcome based on the molecular weight of the analyzed biomolecule. When applied to complex proteoform mixtures in high-throughput top-down proteomics, 213 nm UVPD demonstrated a high degree of complementarity with the most employed fragmentation method in proteomics studies, higher-energy collisional dissociation (HCD). UVPD at 213 nm offered higher average proteoform sequence coverage and degree of proteoform characterization (including localization of post-translational modifications) than HCD. However, previous studies have shown limitations in applying database search strategies developed for HCD fragmentation to UVPD spectra which contains up to nine fragment ion types. We therefore performed an analysis of the different UVPD product ion type frequencies. From these data, we developed an ad hoc fragment matching strategy and determined the influence of each possible ion type on search outcomes. By paring down the number of ion types considered in high-throughput UVPD searches from all types down to the four most abundant, we were ultimately able to achieve deeper proteome characterization with UVPD. Lastly, our detailed product ion analysis also revealed UVPD cleavage propensities and determined the presence of a product ion produced specifically by 213 nm photons. All together, these observations could be used to better elucidate UVPD dissociation mechanisms and improve the utility of the technique for proteomic applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kafader2020,

title = {Native vs Denatured: An in Depth Investigation of Charge State and Isotope Distributions},

author = {Kafader, J.O and Melani, R.D and Schachner, L.F and Ives, A.N and Patrie, S.M and Kelleher, N.L and Compton, P.D},

doi = {10.1021/jasms.9b00040},

year  = {2020},

date = {2020-02-01},

journal = {J Am Soc Mass Spectrom.},

volume = {31},

number = {3},

pages = {574-581},

abstract = {New tools and techniques have dramatically accelerated the field of structural biology over the past several decades. One potent and relatively new technique that is now being utilized by an increasing number of laboratories is the combination of so-called "native" electrospray ionization (ESI) with mass spectrometry (MS) for the characterization of proteins and their noncovalent complexes. However, native ESI-MS produces species at increasingly higher m/z with increasing molecular weight, leading to substantial differences when compared to traditional mass spectrometric approaches using denaturing ESI solutions. Herein, these differences are explored both theoretically and experimentally to understand the role that charge state and isotopic distributions have on signal-to-noise (S/N) as a function of complex molecular weight and how the reduced collisional cross sections of proteins electrosprayed under native solution conditions can lead to improved data quality in image current mass analyzers, such as Orbitrap and FT-ICR. Quantifying ion signal differences under native and denatured conditions revealed enhanced S/N and a more gradual decay in S/N with increasing mass under native conditions. Charge state and isotopic S/N models, supported by experimental results, indicate that analysis of proteins under native conditions at 100 kDa will be 17 times more sensitive than analysis under denatured conditions at the same mass. Higher masses produce even larger sensitivity gains. Furthermore, reduced cross sections under native conditions lead to lower levels of ion decay within an Orbitrap scan event over long transient acquisition times, enabling isotopic resolution of species with molecular weights well in excess of those typically resolved under denatured conditions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Navarro2019,

title = {A computational framework to explore large-scale biosynthetic diversity},

author = {Navarro-Muñoz, J.C and Selem-Mojica, N and Mullowney, M.W and Kautsar, S.A and Tryon, J.H and Parkinson, E.I and De Los Santos, E.L.C and Yeong, M and Cruz-Morales, P and Abubucker, S and Roeters, A and Lokhorst, W and Fernandez-Guerra, A and Cappelini, L.T.D and Goering, A.W and Thomson, R.J and Metcalf, W.W and Kelleher, N.L and Barona-Gomez, F and Medema, M.H},

url = {https://www.nature.com/articles/s41589-019-0400-9},

doi = {10.1038/s41589-019-0400-9},

year  = {2020},

date = {2020-01-01},

journal = {Nat Chem Biol.},

volume = {16},

number = {1},

pages = {60-68},

abstract = {Genome mining has become a key technology to exploit natural product diversity. Although initially performed on a single-genome basis, the process is now being scaled up to mine entire genera, strain collections and microbiomes. However, no bioinformatic framework is currently available for effectively analyzing datasets of this size and complexity. In the present study, a streamlined computational workflow is provided, consisting of two new software tools: the 'biosynthetic gene similarity clustering and prospecting engine' (BiG-SCAPE), which facilitates fast and interactive sequence similarity network analysis of biosynthetic gene clusters and gene cluster families; and the 'core analysis of syntenic orthologues to prioritize natural product gene clusters' (CORASON), which elucidates phylogenetic relationships within and across these families. BiG-SCAPE is validated by correlating its output to metabolomic data across 363 actinobacterial strains and the discovery potential of CORASON is demonstrated by comprehensively mapping biosynthetic diversity across a range of detoxin/rimosamide-related gene cluster families, culminating in the characterization of seven detoxin analogues.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Park2020,

title = {Novel Interface for High-Throughput Analysis of Biotherapeutics by Electrospray Mass Spectrometry},

author = {Park, H.M and Winton, V.J and Drader, J.J and Manalili Wheeler, S and Lazar, G.A and Kelleher, N.L and Liu, Y and Tran, J.C and Compton, P.D},

url = {https://pubs.acs.org/doi/abs/10.1021/acs.analchem.9b04826},

doi = {10.1021/acs.analchem.9b04826},

year  = {2020},

date = {2020-01-01},

journal = {Anal Chem.},

volume = {92},

number = {2},

pages = {2186-2193},

abstract = {With the rapid rise of therapeutic antibodies and antibody-drug conjugates, significant investments have been made in developing workflows that utilize mass spectrometry to detect these intact molecules, the large fragments generated by their selective digestion, and the peptides generated by traditional proteomics workflows. The resultant data is used to gain insight into a wide range of parameters, including primary sequence, disulfide bonding, glycosylation patterns, biotransformation, and more. However, many of the technologies utilized to couple these workflows to mass spectrometers have significant limitations that force nonoptimal modifications to upstream sample preparation steps, limit the throughput of high-volume workflows, and prevent the harmonization of diverse experiments onto a single hardware platform. Here, we describe a new analytical platform that enables direct and high-throughput coupling to electrospray ionization mass spectrometry. The SampleStream platform is compatible with both native and denaturing electrospray, operates with a throughput of up to 15 s/sample, provides extensive concentration of dilute samples, and affords similar sensitivity to comparable liquid chromatographic methods.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Montaser2020,

title = {Discovery of the Biosynthetic Machinery for Stravidins, Biotin Antimetabolites},

author = {Montaser, R and Kelleher, N.L},

url = {https://pubs.acs.org/doi/10.1021/acschembio.9b00890},

doi = {10.1021/acschembio.9b00890},

year  = {2020},

date = {2020-01-01},

journal = {ACS Chem Biol. },

abstract = {Stravidins are peptide antibiotics produced by Streptomyces spp. Their antibacterial activity derives from an unusual amiclenomycin monomer, the warhead that inhibits biotin biosynthesis. Despite being discovered over five decades ago, stravidin biosynthesis has remained a mystery. Using our "metabologenomics" platform, we discover new stravidin analogues and identify the novel biosynthetic machinery responsible for their production. Analysis of the newly identified biosynthetic gene cluster (BGC) indicates the unusual amiclenomycin warhead is derived from chorismic acid, with initial steps similar to those involved in p-amino phenylalanine biosynthesis. However, a distinctive decarboxylation retains the nonaromatic character of a key ring and precedes a one-carbon extension to afford the warhead in its bioactive, untriggered state. Strikingly, we also identified two streptavidin genes flanking the new stravidin BGC reported here. This aligns with the known synergistic activity between the biotin-binding activity of streptavidin and the stravidins to antagonize both biotin biogenesis and bacterial growth.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Winer2020,

title = {Identification of Plasmodium falciparum proteoforms from liver stage models},

author = {Winer, B and Edgel, K.A and Zou, X and Sellau, J and Hadiwidjojo, S and Garver, L.S and McDonough, C.E and Kelleher, N.L and Thomas, P.M and Villasante, E and Ploss, A and Gerbasi, V.R},

url = {https://malariajournal.biomedcentral.com/articles/10.1186/s12936-019-3093-3},

doi = {10.1186/s12936-019-3093-3},

year  = {2020},

date = {2020-01-01},

journal = {Malar J.},

volume = {19},

number = {1},

pages = {10},

abstract = {Immunization with attenuated malaria sporozoites protects humans from experimental malaria challenge by mosquito bite. Protection in humans is strongly correlated with the production of T cells targeting a heterogeneous population of pre-erythrocyte antigen proteoforms, including liver stage antigens. Currently, few T cell epitopes derived from Plasmodium falciparum, the major aetiologic agent of malaria in humans are known.



METHODS:

In this study both in vitro and in vivo malaria liver stage models were used to sequence host and pathogen proteoforms. Proteoforms from these diverse models were subjected to mild acid elution (of soluble forms), multi-dimensional fractionation, tandem mass spectrometry, and top-down bioinformatics analysis to identify proteoforms in their intact state.



RESULTS:

These results identify a group of host and malaria liver stage proteoforms that meet a 5% false discovery rate threshold.



CONCLUSIONS:

This work provides proof-of-concept for the validity of this mass spectrometry/bioinformatic approach for future studies seeking to reveal malaria liver stage antigens towards vaccine development.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Doubleday2020,

title = {Elucidating Proteoform Dynamics Underlying the Senescence Associated Secretory Phenotype},

author = {Doubleday, P.F and Fornelli, L and Kelleher, N.L},

url = {https://pubs.acs.org/doi/10.1021/acs.jproteome.9b00739},

doi = {10.1021/acs.jproteome.9b00739},

year  = {2020},

date = {2020-01-01},

journal = {J Proteome Res.},

abstract = {Primary diploid cells exit the cell cycle in response to exogenous stress or oncogene activation through a process known as cellular senescence. This cell-autonomous tumor-suppressive mechanism is also a major mechanism operative in organismal aging. To date, temporal aspects of senescence remain understudied. Therefore, we use quantitative proteomics to investigate changes following forced HRASG12V expression and induction of senescence across 1 week in normal diploid fibroblasts. We demonstrate that global intracellular proteomic changes correlate with the emergence of the senescence-associated secretory phenotype and the switch to robust cell cycle exit. The senescence secretome reinforces cell cycle exit, yet is largely detrimental to tissue homeostasis. Previous studies of secretomes rely on ELISA, bottom-up proteomics or RNA-seq. To date, no study to date has examined the proteoform complexity of secretomes to elucidate isoform-specific, post-translational modifications or regulated cleavage of signal peptides. Therefore, we use a quantitative top-down proteomics approach to define the molecular complexity of secreted proteins <30 kDa. We identify multiple forms of immune regulators with known activities and affinities such as distinct forms of interleukin-8, as well as GROα and HMGA1, and temporally resolve secreted proteoform dynamics. Together, our work demonstrates the complexity of the secretome past individual protein accessions and provides motivation for further proteoform-resolved measurements of the secretome.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Navarro-Munoz2019,

title = {A computational framework to explore large-scale biosynthetic diversity},

author = {Navarro-Muñoz, J.C and Selem-Mojica, N and Mullowney, M.W and Kautsar, S.A and Tryon, J.H and Parkinson, E.I and De Los Santos, E.L.C and Yeong, M and Cruz-Morales, P and Abubucker, S and Roeters, A and Lokhorst, W and Fernandez-Guerra, A and Cappelini, L.T.D and Goering, A.W and Thomson, R.J and Metcalf, W.W and Kelleher, N.L and Barona-Gomez, F and Medema, M.H. },

url = {https://www.nature.com/articles/s41589-019-0400-9},

doi = {10.1038/s41589-019-0400-9},

year  = {2020},

date = {2020-01-01},

journal = {Nat Chem Biol.},

volume = {16},

number = {10},

pages = {60-68},

abstract = {Genome mining has become a key technology to exploit natural product diversity. Although initially performed on a single-genome basis, the process is now being scaled up to mine entire genera, strain collections and microbiomes. However, no bioinformatic framework is currently available for effectively analyzing datasets of this size and complexity. In the present study, a streamlined computational workflow is provided, consisting of two new software tools: the 'biosynthetic gene similarity clustering and prospecting engine' (BiG-SCAPE), which facilitates fast and interactive sequence similarity network analysis of biosynthetic gene clusters and gene cluster families; and the 'core analysis of syntenic orthologues to prioritize natural product gene clusters' (CORASON), which elucidates phylogenetic relationships within and across these families. BiG-SCAPE is validated by correlating its output to metabolomic data across 363 actinobacterial strains and the discovery potential of CORASON is demonstrated by comprehensively mapping biosynthetic diversity across a range of detoxin/rimosamide-related gene cluster families, culminating in the characterization of seven detoxin analogues.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Camarillo2019,

title = {Coupling Fluorescence-Activated Cell Sorting and Targeted Analysis of Histone Modification Profiles in Primary Human Leukocytes},

author = {Camarillo, J.M and Swaminathan, S and Abshiru, N.A and Sikora, J.W and Thomas, P.M and Kelleher, N.L},

url = {https://www.ncbi.nlm.nih.gov/pubmed/31286445},

doi = {10.1007/s13361-019-02255-x},

year  = {2019},

date = {2019-12-01},

journal = {J Am Soc Mass Spectrom.},

volume = {30},

number = {12},

pages = {2526-2534},

abstract = {Histone posttranslational modifications (PTMs) are essential for regulating chromatin and maintaining gene expression throughout cell differentiation. Despite the deep level of understanding of immunophenotypic differentiation pathways in hematopoietic cells, few studies have investigated global levels of histone PTMs required for differentiation and maintenance of these distinct cell types. Here, we describe an approach to couple fluorescence-activated cell sorting (FACS) with targeted mass spectrometry to define global "epi-proteomic" signatures for primary leukocytes. FACS was used to sort closely and distantly related leukocytes from normal human peripheral blood for quantitation of histone PTMs with a multiple reaction monitoring LC-MS/MS method measuring histone PTMs on histones H3 and H4. We validate cell sorting directly into H2SO4 for immediate histone extraction to decrease time and number of steps after FACS to analyze histone PTMs. Relative histone PTM levels vary in T cells across healthy donors, and the majority of PTMs remain stable up to 2 days following initial blood draw. Large differences in the levels of histone PTMs are observed across the mature lymphoid and myeloid lineages, as well as between different types within the same lineage, though no differences are observed in closely related T cell subtypes. The results show a streamlined approach for quantifying global changes in histone PTMs in cell types separated by FACS that is poised for clinical deployment.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Melani2019,

title = {Direct measurement of light and heavy antibody chains using ion mobility and middle-down mass spectrometry},

author = {Melani, R.D and Srzentić, K and Gerbasi, V.R and McGee, J.P and Huguet, R and Fornelli, L and Kelleher, N.L},

url = {https://www.tandfonline.com/doi/full/10.1080/19420862.2019.1668226},

doi = {10.1080/19420862.2019.1668226},

year  = {2019},

date = {2019-12-01},

journal = {MAbs.},

volume = {11},

number = {8},

pages = {1351-1357},

abstract = {The analysis of monoclonal antibodies (mAbs) by a middle-down mass spectrometry (MS) approach is a growing field that attracts the attention of many researchers and biopharmaceutical companies. Usually, liquid fractionation techniques are used to separate mAbs polypeptides chains before MS analysis. Gas-phase fractionation techniques such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) can replace liquid-based separations and reduce both analysis time and cost. Here, we present a rapid FAIMS tandem MS method capable of characterizing the polypeptide sequence of mAbs light and heavy chains in an unprecedented, easy, and fast fashion. This new method uses commercially available instruments and takes ~24 min, which is 40-60% faster than regular liquid chromatography-MS/MS analysis, to acquire fragmentation data using different dissociation methods.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{DesSoye2019,

title = {A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli},

author = {Des Soye, B.J and Gerbasi, V.R and Thomas, P.M and Kelleher, N.L and Jewett, M.C},

url = {https://www.sciencedirect.com/science/article/pii/S2451945619303538?via%3Dihub},

doi = {10.1016/j.chembiol.2019.10.008},

year  = {2019},

date = {2019-12-01},

journal = {Cell Chem Biol.},

volume = {26},

number = {12},

pages = {1743-1754},

abstract = {The site-specific incorporation of non-canonical amino acids (ncAAs) into proteins via amber suppression provides access to novel protein properties, structures, and functions. Historically, poor protein expression yields resulting from release factor 1 (RF1) competition has limited this technology. To address this limitation, we develop a high-yield, one-pot cell-free platform for synthesizing proteins bearing ncAAs based on genomically recoded Escherichia coli lacking RF1. A key feature of this platform is the independence on the addition of purified T7 DNA-directed RNA polymerase (T7RNAP) to catalyze transcription. Extracts derived from our final strain demonstrate high productivity, synthesizing 2.67 ± 0.06 g/L superfolder GFP in batch mode without supplementation of purified T7RNAP. Using an optimized one-pot platform, we demonstrate multi-site incorporation of the ncAA p-acetyl-L-phenylalanine into an elastin-like polypeptide with high accuracy of incorporation and yield. Our work has implications for chemical and synthetic biology.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Huguet2019,

title = {Proton Transfer Charge Reduction Enables High-Throughput Top-Down Analysis of Large Proteoforms},

author = {Huguet, R and Mullen, C and Srzentić, K and Greer, J.B and Fellers, R.T and Zabrouskov, V and Syka, J.E.P and Kelleher, N.L and Fornelli, L},

url = {https://pubs.acs.org/doi/abs/10.1021/acs.analchem.9b03925},

doi = {10.1021/acs.analchem.9b03925},

year  = {2019},

date = {2019-12-01},

journal = {Anal Chem.},

volume = {91},

number = {24},

pages = {15732-15739},

abstract = {Despite the recent technological advances in Fourier transform mass spectrometry (FTMS) instrumentation, top-down proteomics (TDP) is currently mostly applied to the characterization of proteoforms <30 kDa due to the poor performance of high-resolution FTMS for the analysis of larger proteoforms and the high complexity of intact proteomes in the 30-60 kDa mass range. Here, we propose a novel data acquisition method based on ion-ion proton transfer, herein termed proton transfer charge reduction (PTCR), to investigate large proteoforms of Pseudomonas aeruginosa in a high-throughput fashion. We designed a targeted data acquisition strategy, named tPTCR, which applies two consecutive gas phase fractionation steps for obtaining intact precursor masses: first, a narrow (1.5 m/z-wide) quadrupole filter m/z transmission window is used to select a subset of charge states from all ionized proteoform cations; second, this aliquot of protein cations is subjected to PTCR in order to reduce their average charge state: upon m/z analysis in an Orbitrap, proteoform mass spectra with minimal m/z peak overlap and easy-to-interpret charge state distributions are obtained, simplifying the proteoform mass calculation. Subsequently, the same quadrupole-selected narrow m/z region of analytes is subjected to collisional dissociation to obtain proteoform sequence information, which used in combination with intact mass information leads to proteoform identification through an off-line database search. The newly proposed method was benchmarked against the previously developed "medium/high" data-dependent acquisition strategy and doubled the number of UniProt entries and proteoforms >30 kDa identified on the liquid chromatography time scale.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ling2019,

title = {Chromatin occupancy and epigenetic analysis reveal new insights into the function of the GATA1 N terminus in erythropoiesis},

author = {Ling, T and Birger, Y and Stankiewicz, M J and Ben-Haim, N and Kalisky, T and Rein, A and Kugler, E and Chen, W and Fu, C and Zhang, K and Patel, H and Sikora, J W and Goo, Y A and Kelleher, N and Zou, L and Izraeli, S and Crispino, J D },

url = {https://ashpublications.org/blood/article/134/19/1619/374957/Chromatin-occupancy-and-epigenetic-analysis-reveal},

doi = {10.1182/blood.2019001234},

year  = {2019},

date = {2019-11-07},

journal = {Blood},

volume = {134},

number = {19},

pages = {1619 - 1631},

abstract = {Mutations in GATA1, which lead to expression of the GATA1s isoform that lacks the GATA1 N terminus, are seen in patients with Diamond-Blackfan anemia (DBA). In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in Gata1s mice. Defects in yolks sac and fetal liver hematopoiesis included impaired terminal maturation and reduced numbers of erythroid progenitors. RNA-sequencing revealed that both erythroid and megakaryocytic gene expression patterns were altered by the loss of the N terminus, including aberrant upregulation of Gata2 and Runx1. Dysregulation of global H3K27 methylation was found in the erythroid progenitors upon loss of N terminus of GATA1. Chromatin-binding assays revealed that, despite similar occupancy of GATA1 and GATA1s, there was a striking reduction of H3K27me3 at regulatory elements of the Gata2 and Runx1 genes. Consistent with the observation that overexpression of GATA2 has been reported to impair erythropoiesis, we found that haploinsufficiency of Gata2 rescued the erythroid defects of Gata1s fetuses. Together, our integrated genomic analysis of transcriptomic and epigenetic signatures reveals that, Gata1 mice provide novel insights into the role of the N terminus of GATA1 in transcriptional regulation and red blood cell maturation which may potentially be useful for DBA patients.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kafader2019b,

title = {STORI Plots Enable Accurate Tracking of Individual Ion Signals},

author = {Kafader, J.O and Beu, S.C and Early, B.P and Melani, R.D and Durbin, K.R and Zabrouskov, V and Makarov, A.A and Maze, J.T and Shinholt, D.L and Yip, P.F and Kelleher, N.L and Compton, P.D and Senko, M.W},

url = {https://www.ncbi.nlm.nih.gov/pubmed/31512223},

doi = {10.1007/s13361-019-02309-0},

year  = {2019},

date = {2019-11-01},

journal = {J Am Soc Mass Spectrom.},

volume = {30},

number = {11},

pages = {2200-2203},

abstract = {Charge detection mass spectrometry (CDMS) of low-level signals is currently limited to the analysis of individual ions that generate a persistent signal during the entire observation period. Ions that disintegrate during the observation period produce reduced frequency domain signal amplitudes, which lead to an underestimation of the ion charge state, and thus the ion mass. The charge assignment can only be corrected through an accurate determination of the time of ion disintegration. The traditional mechanisms for temporal signal analysis have severe limitations for temporal resolution, spectral resolution, and signal-to-noise ratios. Selective Temporal Overview of Resonant Ions (STORI) plots provide a new framework to accurately analyze low-level time domain signals of individual ions. STORI plots allow for complete correction of intermittent signals, the differentiation of single and multiple ions at the same frequency, and the association of signals that spontaneously change frequency.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Park2019,

title = {Multidimensional Top-Down Proteomics of Brain-Region-Specific Mouse Brain Proteoforms Responsive to Cocaine and Estradiol},

author = {Park, H.M and Satta, R and Davis, R.G and Goo, Y.A and LeDuc, R.D and Fellers, R.T and Greer, J.B and Romanova, E.V and Rubakhin, S.S and Tai, R and Thomas, P.M and Sweedler, J.V and Kelleher, N.L and Patrie, S.M and Lasek, A.W},

url = {https://pubs.acs.org/doi/10.1021/acs.jproteome.9b00481},

doi = {10.1021/acs.jproteome.9b00481},

year  = {2019},

date = {2019-11-01},

journal = {J Proteome Res.},

volume = {18},

number = {11},

pages = {3999-4012},

abstract = {Cocaine addiction afflicts nearly 1 million adults in the United States, and to date, there are no known treatments approved for this psychiatric condition. Women are particularly vulnerable to developing a cocaine use disorder and suffer from more serious cardiac consequences than men when using cocaine. Estrogen is one biological factor contributing to the increased risk for females to develop problematic cocaine use. Animal studies have demonstrated that estrogen (17β-estradiol or E2) enhances the rewarding properties of cocaine. Although E2 affects the dopamine system, the molecular and cellular mechanisms of E2-enhanced cocaine reward have not been characterized. In this study, quantitative top-down proteomics was used to measure intact proteins in specific regions of the female mouse brain after mice were trained for cocaine-conditioned place preference, a behavioral test of cocaine reward. Several proteoform changes occurred in the ventral tegmental area after combined cocaine and E2 treatments, with the most numerous proteoform alterations on myelin basic protein, indicating possible changes in white matter structure. There were also changes in histone H4, protein phosphatase inhibitors, cholecystokinin, and calmodulin proteoforms. These observations provide insight into estrogen signaling in the brain and may guide new approaches to treating women with cocaine use disorder.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Schneider2019,

title = {A MUC16 IgG binding activity selects for a restricted subset of IgG enriched for certain SIV epitope specificities},

author = {Schneider, J.R and Shen, X and Orlandi, C and Nyanhete, T and Sawant, S and Carias, A.M and Smith, A.D and Kelleher, N.L and Veazey, R.S and Lewis, G.K and Tomaras, G.D and Hope, T.J},

url = {https://jvi.asm.org/content/early/2019/11/21/JVI.01246-19.long},

doi = {10.1128/JVI.01246-19},

year  = {2019},

date = {2019-11-01},

journal = {J Virol.},

abstract = {We have recently shown that MUC16, a component of the glycocalyx of some mucosal barriers, has elevated binding to the G0 glycoform of the Fc portion of IgG. Therefore, IgG from chronically infected HIV patients, who typically exhibit increased amounts of G0 glycoforms, showed increased MUC16 binding compared to uninfected controls. Using the rhesus macaque SIVmac251 model, we can compare plasma antibodies before and after chronic infection. We find increased binding of IgG to MUC16 after chronic SIV infection. Antibodies isolated for tight association with MUC16 (MUC16-eluted antibodies) show reduced FcγR engagement and antibody-dependent cellular cytotoxicity (ADCC) activity. The glycosylation profile of these IgGs was consistent with a decrease in FcγR engagement and subsequent ADCC effector function as they contain a decrease in afucosylated bisecting glycoforms that preferentially bind FcγRs. Testing of the SIV antigen specificity of IgG from SIV infected macaques revealed the MUC16-eluted antibodies were enriched for certain specific epitopes including regions of gp41 and gp120. This enrichment of specific antigen responses for fucosylated bisecting glycoforms and subsequent association with MUC16 suggests the immune response has the potential to direct specific epitope responses to localize to the glycocalyx through interaction with this specific mucin.Importance Understanding how antibodies are distributed in the mucosal environment is valuable for development of a vaccine to block HIV infection. Here we study an IgG binding activity in MUC16, potentially representing a new IgG effector function that would concentrate certain antibodies within the glycocalyx to trap pathogens before they can reach the underlying columnar epithelial barriers. These studies reveal that rhesus macaque IgG responses during chronic SIV infection generate increased antibodies that bind MUC16 and interestingly these MUC16-tethered antibodies are enriched for binding to certain antigens. Therefore, it may be possible to direct HIV vaccine-generated responses to associate with MUC16 and enhance the antibody's ability to mediate immune exclusion by trapping virions within the glycocalyx and preventing the virus from reaching immune target cells within the mucosa. This concept will ultimately have to be tested in the rhesus macaque model which is shown here to have MUC16 targeted antigen responses.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Bennett2019,

title = {A Mutation in Histone H2B Represents a New Class of Oncogenic Driver},

author = {Bennett, R.L and Bele, A and Small, E.C and Will, C.M and Nabet, B and Oyer, J.A and Huang, X and Ghosh, R.P and Grzybowski, A.T and Yu, T and Zhang, Q and Riva, A and Lele, T.P and Schatz, G.C and Kelleher, N.L and Ruthenburg, A.J and Liphardt, J and Licht, J.D},

url = {http://cancerdiscovery.aacrjournals.org/cgi/pmidlookup?view=long&pmid=31337617},

doi = {10.1158/2159-8290.CD-19-0393},

year  = {2019},

date = {2019-10-01},

journal = {Cancer Discov.},

volume = {9},

number = {10},

pages = {1438-1451},

abstract = {By examination of the cancer genomics database, we identified a new set of mutations in core histones that frequently recur in cancer patient samples and are predicted to disrupt nucleosome stability. In support of this idea, we characterized a glutamate to lysine mutation of histone H2B at amino acid 76 (H2B-E76K), found particularly in bladder and head and neck cancers, that disrupts the interaction between H2B and H4. Although H2B-E76K forms dimers with H2A, it does not form stable histone octamers with H3 and H4 in vitro, and when reconstituted with DNA forms unstable nucleosomes with increased sensitivity to nuclease. Expression of the equivalent H2B mutant in yeast restricted growth at high temperature and led to defective nucleosome-mediated gene repression. Significantly, H2B-E76K expression in the normal mammary epithelial cell line MCF10A increased cellular proliferation, cooperated with mutant PIK3CA to promote colony formation, and caused a significant drift in gene expression and fundamental changes in chromatin accessibility, particularly at gene regulatory elements. Taken together, these data demonstrate that mutations in the globular domains of core histones may give rise to an oncogenic program due to nucleosome dysfunction and deregulation of gene expression. SIGNIFICANCE: Mutations in the core histones frequently occur in cancer and represent a new mechanism of epigenetic dysfunction that involves destabilization of the nucleosome, deregulation of chromatin accessibility, and alteration of gene expression to drive cellular transformation.See related commentary by Sarthy and Henikoff, p. 1346.This article is highlighted in the In This Issue feature, p. 1325.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Smith2019,

title = {A five-level classification system for proteoform identifications},

author = {Smith, L.M and Thomas, P.M and Shortreed, M.R and Schaffer, L.V and Fellers, R.T and LeDuc, R.D and Tucholski,, T and Ge, Y and Agar, J.N and Anderson, L.C and Chamot-Rooke, J and Gault, J and Loo, J.A and Paša-Tolić, L and Robinson, C.V and Schlüter, H and Tsybin, Y.O and Vilaseca, M and Vizcaíno, J.A and Danis, P.O and Kelleher, N.L},

url = {https://www.nature.com/articles/s41592-019-0573-x},

doi = {10.1038/s41592-019-0573-x},

year  = {2019},

date = {2019-10-01},

journal = {Nat Methods.},

volume = {16},

number = {10},

pages = {939-940},

abstract = {The term proteoform, introduced in Nature Methods in 2013 (ref. 1), has rapidly gained acceptance in the proteomics community. The challenge and importance of comprehensively identifying proteoforms in complex samples has been recognized, and reports have begun to appear of new platforms towards that end2,3,4,5. However, one interesting central ambiguity has emerged, namely determining precisely what is meant by a ‘proteoform identification’. At present, the only practical approaches for establishing the exact primary structure of a proteoform employ mass spectrometry (MS), and a wide range of MS results claim proteoform identifications6. This seemingly small matter has significant impact, as the ambiguity in what is meant by an ‘identification’ makes it difficult to compare results from different laboratories and approaches. This situation hinders the ability of the community to evaluate technological progress and to efficiently expand biological knowledge.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Lue2019,

title = {Precision Targeting with EZH2 and HDAC Inhibitors in Epigenetically Dysregulated Lymphomas},

author = {Lue, J.K and Prabhu, S.A and Liu, Y and Gonzalez, Y and Verma, A and Mundi, P.S and Abshiru, N and Camarillo, J.M and Mehta, S and Chen, E.I and Qiao, C and Nandakumar, R and Cremers, S and Kelleher, N.L and Elemento, O and Amengual, J.E},

url = {https://clincancerres.aacrjournals.org/content/25/17/5271.long},

doi = {10.1158/1078-0432.CCR-18-3989},

year  = {2019},

date = {2019-09-01},

journal = {Clin Cancer Res},

volume = {25},

number = {17},

pages = {5271-5283},

abstract = {PURPOSE:

Both gain-of-function enhancer of zeste homolog 2 (EZH2) mutations and inactivating histone acetyltransferases mutations, such as CREBBP and EP300, have been implicated in the pathogenesis of germinal center (GC)-derived lymphomas. We hypothesized that direct inhibition of EZH2 and histone deacetyltransferase (HDAC) would be synergistic in GC-derived lymphomas.



EXPERIMENTAL DESIGN:

Lymphoma cell lines (n = 21) were exposed to GSK126, an EZH2 inhibitor, and romidepsin, a pan-HDAC inhibitor. Synergy was assessed by excess over bliss. Western blot, mass spectrometry, and coimmunoprecipitation were performed. A SU-DHL-10 xenograft model was utilized to validate in vitro findings. Pretreatment RNA-sequencing of cell lines was performed. MetaVIPER analysis was used to infer protein activity.



RESULTS:

Exposure to GSK126 and romidepsin demonstrated potent synergy in lymphoma cell lines with EZH2 dysregulation. Combination of romidepsin with other EZH2 inhibitors also demonstrated synergy suggesting a class effect of EZH2 inhibition with romidepsin. Dual inhibition of EZH2 and HDAC led to modulation of acetylation and methylation of H3K27. The synergistic effects of the combination were due to disruption of the PRC2 complex secondary to acetylation of RbAP 46/48. A common basal gene signature was shared among synergistic lymphoma cell lines and was characterized by upregulation in chromatin remodeling genes and transcriptional regulators. This finding was supported by metaVIPER analysis which also revealed that HDAC 1/2 and DNA methyltransferase were associated with EZH2 activation.



CONCLUSIONS:

Inhibition of EZH2 and HDAC is synergistic and leads to the dissociation of PRC2 complex. Our findings support the clinical translation of the combination of EZH2 and HDAC inhibition in EZH2 dysregulated lymphomas.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Huang2019,

title = {Defining the NSD2 interactome: PARP1 PARylation reduces NSD2 histone methyltransferase activity and impedes chromatin binding},

author = {Huang, X and LeDuc, R.D and Fornelli, L and Schunter, A.J and Bennett, R.L and Kelleher, N.L and Licht, J.D},

url = {https://www.jbc.org/content/294/33/12459.long},

doi = {10.1074/jbc.RA118.006159},

year  = {2019},

date = {2019-08-01},

journal = {J Biol Chem.},

volume = {294},

number = {33},

pages = {12459-12471},

abstract = {NSD2 is a histone methyltransferase that specifically dimethylates histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. Dramatic overexpression of NSD2 in t(4;14) multiple myeloma (MM) and an activating mutation of NSD2 discovered in acute lymphoblastic leukemia are significantly associated with altered gene activation, transcription, and DNA damage repair. The partner proteins through which NSD2 may influence critical cellular processes remain poorly defined. In this study, we utilized proximity-based labeling (BioID) combined with label-free quantitative MS to identify high confidence NSD2 interacting partners in MM cells. The top 24 proteins identified were involved in maintaining chromatin structure, transcriptional regulation, RNA pre-spliceosome assembly, and DNA damage. Among these, an important DNA damage regulator, poly(ADP-ribose) polymerase 1 (PARP1), was discovered. PARP1 and NSD2 have been found to be recruited to DNA double strand breaks upon damage and H3K36me2 marks are enriched at damage sites. We demonstrate that PARP1 regulates NSD2 via PARylation upon oxidative stress. In vitro assays suggest the PARylation significantly reduces NSD2 histone methyltransferase activity. Furthermore, PARylation of NSD2 inhibits its ability to bind to nucleosomes and further get recruited at NSD2-regulated genes, suggesting PARP1 regulates NSD2 localization and H3K36me2 balance. This work provides clear evidence of cross-talk between PARylation and histone methylation and offers new directions to characterize NSD2 function in DNA damage response, transcriptional regulation, and other pathways.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Schachner2019,

title = {Standard Proteoforms and Their Complexes for Native Mass Spectrometry},

author = {Schachner, L.F and Ives, A.N and McGee, J.P and Melani, R.D and Kafader, J.O and Compton, P.D and Patrie, S.M and Kelleher, N.L},

doi = {10.1007/s13361-019-02191-w},

year  = {2019},

date = {2019-07-01},

journal = {Journal of the American Society for Mass Spectrometry},

volume = {30},

number = {7},

pages = {1190-1198},

abstract = {Native mass spectrometry (nMS) is a technique growing at the interface of analytical chemistry, structural biology, and proteomics that enables the detection and partial characterization of non-covalent protein assemblies. Currently, the standardization and dissemination of nMS is hampered by technical challenges associated with instrument operation, benchmarking, and optimization over time. Here, we provide a standard operating procedure for acquiring high-quality native mass spectra of 30-300 kDa proteins using an Orbitrap mass spectrometer. By describing reproducible sample preparation, loading, ionization, and nMS analysis, we forward two proteoforms and three complexes as possible standards to advance training and longitudinal assessment of instrument performance. Spectral data for five standards can guide assessment of instrument parameters, data production, and data analysis. By introducing this set of standards and protocols, we aim to help normalize native mass spectrometry practices across labs and provide benchmarks for reproducibility and high-quality data production in the years ahead. Graphical abstract.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Donnelly2019,

title = {Best practices and benchmarks for intact protein analysis for top-down mass spectrometry},

author = {Donnelly, D.P and Rawlins, C.M and DeHart, C.J and Fornelli, L and Schachner, L.F and Lin, Z and Lippens, J.L and Aluri, K.C and Sarin, R and Chen, B and Lantz, C and Jung, W and Johnson, K.R and Koller, A and Wolff, J.J and Campuzano, I.D.G and Auclair, J.R and Ivanov, A.R and Whitelegge, J.P and Paša-Tolić, L and Chamot-Rooke, J and Danis, P.O and Smith, L.M and Tsybin, Y.O and Loo, J.A and Ge, Y and Kelleher, N.L and Agar, J.N},

url = {https://www.nature.com/articles/s41592-019-0457-0},

doi = {10.1038/s41592-019-0457-0},

year  = {2019},

date = {2019-07-01},

journal = {Nat Methods.},

volume = {16},

number = {7},

pages = {587-594},

abstract = {One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Moschitto2019,

title = {Mechanism of Inactivation of Ornithine Aminotransferase by (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)cyclopentane-1-carboxylic Acid},

author = {Moschitto, M.J and Doubleday, P.F and Catlin, D.S and Kelleher, N.L and Liu, D and Silverman, R.B},

url = {https://pubs.acs.org/doi/10.1021/jacs.9b03254},

doi = {10.1021/jacs.9b03254},

year  = {2019},

date = {2019-07-01},

journal = {J Am Chem Soc.},

volume = {14},

number = {27},

pages = {10711-10721},

abstract = {The inhibition of ornithine aminotransferase (OAT), a pyridoxal 5'-phosphate-dependent enzyme, has been implicated as a treatment for hepatocellular carcinoma (HCC), the most common form of liver cancer, for which there is no effective treatment. From a previous evaluation of our aminotransferase inhibitors, (1S,3S)-3-amino-4-(perfluoropropan-2-ylidene)cyclopentane-1-carboxylic acid hydrochloride (1) was found to be a selective and potent inactivator of human OAT (hOAT), which inhibited the growth of HCC in athymic mice implanted with human-derived HCC, even at a dose of 0.1 mg/kg. Currently, investigational new drug (IND)-enabling studies with 1 are underway. The inactivation mechanism of 1, however, has proved to be elusive. Here we propose three possible mechanisms, based on mechanisms of known aminotransferase inactivators: Michael addition, enamine addition, and fluoride ion elimination followed by conjugate addition. On the basis of crystallography and intact protein mass spectrometry, it was determined that 1 inactivates hOAT through fluoride ion elimination to an activated 1,1'-difluoroolefin, followed by conjugate addition and hydrolysis. This result was confirmed with additional studies, including the detection of the cofactor structure by mass spectrometry and through the identification of turnover metabolites. On the basis of this inactivation mechanism and to provide further evidence for the mechanism, analogues of 1 (19, 20) were designed, synthesized, and demonstrated to have the predicted selective inactivation mechanism. These analogues highlight the importance of the trifluoromethyl group and provide a basis for future inactivator design.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ro2019,

title = {Native top-down mass spectrometry provides insights into the copper centers of membrane-bound methane monooxygenase},

author = {Ro, S.Y and Schachner, L.F and Koo, C.W and Purohit, R and Remis, J.P and Kenney, G.E and Liauw, B.W and Thomas, P.M and Patrie, S.M and Kelleher, N.L and Rosenzweig, A.C },

url = {https://www.nature.com/articles/s41467-019-10590-6},

doi = {10.1038/s41467-019-10590-6},

year  = {2019},

date = {2019-06-01},

journal = {Nat Commun.},

volume = {10},

number = {1},

pages = {2675},

abstract = {Aerobic methane oxidation is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent, membrane metalloenzyme composed of subunits PmoA, PmoB, and PmoC. Characterization of the copper active site has been limited by challenges in spectroscopic analysis stemming from the presence of multiple copper binding sites, effects of detergent solubilization on activity and crystal structures, and the lack of a heterologous expression system. Here we utilize nanodiscs coupled with native top-down mass spectrometry (nTDMS) to determine the copper stoichiometry in each pMMO subunit and to detect post-translational modifications (PTMs). These results indicate the presence of a mononuclear copper center in both PmoB and PmoC. pMMO-nanodisc complexes with a higher stoichiometry of copper-bound PmoC exhibit increased activity, suggesting that the PmoC copper site plays a role in methane oxidation activity. These results provide key insights into the pMMO copper centers and demonstrate the ability of nTDMS to characterize complex membrane-bound metalloenzymes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Schaffer2019,

title = {Identification and Quantification of Proteoforms by Mass Spectrometry},

author = {Schaffer, L.V and Millikin, R.J and Miller, R.M and Anderson, L.C and Fellers, R.T and Ge, Y and Kelleher, N.L and LeDuc, R.D and Liu, X and Payne, S.H and Sun, L and Thomas, P.M and Tucholski, T and Wang, Z and Wu, S and Wu, Z and Yu, D and Shortreed, M.R and Smith, L.M},

url = {https://onlinelibrary.wiley.com/doi/full/10.1002/pmic.201800361},

doi = {10.1002/pmic.201800361},

year  = {2019},

date = {2019-05-01},

journal = {Proteomics and Systems Biology},

volume = {19},

number = {10},

abstract = {A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post-translational modifications. In top-down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top-down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{LeDuc2019,

title = {Accurate Estimation of Context-Dependent False Discovery Rates in Top-Down Proteomics},

author = {LeDuc, R.D and Fellers, R.T and Early, B.P and Greer, J.B and Shams, D.P and Thomas, P and Kelleher, N.L. },

url = {https://www.mcponline.org/content/18/4/796.long},

doi = {10.1074/mcp.RA118.000993},

year  = {2019},

date = {2019-04-01},

journal = {Mol Cell Proteomics.},

volume = {18},

number = {4},

pages = {796-805},

abstract = {Within the last several years, top-down proteomics has emerged as a high throughput technique for protein and proteoform identification. This technique has the potential to identify and characterize thousands of proteoforms within a single study, but the absence of accurate false discovery rate (FDR) estimation could hinder the adoption and consistency of top-down proteomics in the future. In automated identification and characterization of proteoforms, FDR calculation strongly depends on the context of the search. The context includes MS data quality, the database being interrogated, the search engine, and the parameters of the search. Particular to top-down proteomics-there are four molecular levels of study: proteoform spectral match (PrSM), protein, isoform, and proteoform. Here, a context-dependent framework for calculating an accurate FDR at each level was designed, implemented, and validated against a manually curated training set with 546 confirmed proteoforms. We examined several search contexts and found that an FDR calculated at the PrSM level under-reported the true FDR at the protein level by an average of 24-fold. We present a new open-source tool, the TDCD_FDR_Calculator, which provides a scalable, context-dependent FDR calculation that can be applied post-search to enhance the quality of results in top-down proteomics from any search engine.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Cline2018,

title = {A novel crosslinking protocol stabilizes amyloid β oligomers capable of inducing Alzheimer's-associated pathologies},

author = {Cline, E.N and Das, A and Bicca, M.A and Mohammad, S.N and Schachner, L.F and Kamel, J.M and DiNunno, N and Weng, A and Paschall, J.D and Bu, R.L and Khan, F.M and Rollins, M.G and Ives, A.N and Shekhawat, G and Nunes-Tavares, N and de Mello, F.G and Compton, P.D and Kelleher, N.L and Klein, W.L. },

doi = {10.1111/jnc.14647},

year  = {2019},

date = {2019-03-01},

journal = {J Neurochem.},

volume = {148},

number = {6},

pages = {822-836},

abstract = {Amyloid β oligomers (AβOs) accumulate early in Alzheimer's disease (AD) and experimentally cause memory dysfunction and the major pathologies associated with AD, for example, tau abnormalities, synapse loss, oxidative damage, and cognitive dysfunction. In order to develop the most effective AβO-targeting diagnostics and therapeutics, the AβO structures contributing to AD-associated toxicity must be elucidated. Here, we investigate the structural properties and pathogenic relevance of AβOs stabilized by the bifunctional crosslinker 1,5-difluoro-2,4-dinitrobenzene (DFDNB). We find that DFDNB stabilizes synthetic Aβ in a soluble oligomeric conformation. With DFDNB, solutions of Aβ that would otherwise convert to large aggregates instead yield solutions of stable AβOs, predominantly in the 50-300 kDa range, that are maintained for at least 12 days at 37°C. Structures were determined by biochemical and native top-down mass spectrometry analyses. Assayed in neuronal cultures and i.c.v.-injected mice, the DFDNB-stabilized AβOs were found to induce tau hyperphosphorylation, inhibit choline acetyltransferase, and provoke neuroinflammation. Most interestingly, DFDNB crosslinking was found to stabilize an AβO conformation particularly potent in inducing memory dysfunction in mice. Taken together, these data support the utility of DFDNB crosslinking as a tool for stabilizing pathogenic AβOs in structure-function studies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kafader2019,

title = {Measurement of Individual Ions Sharply Increases the Resolution of Orbitrap Mass Spectra of Proteins},

author = {Kafader, J.O and Melani, R.D and Senko, M.W and Makarov, A.A and Kelleher, N.L and Compton, P.D. },

url = {https://pubs.acs.org/doi/10.1021/acs.analchem.8b04519},

doi = {10.1021/acs.analchem.8b04519},

year  = {2019},

date = {2019-02-19},

journal = {Anal Chem.},

volume = {91},

number = {4},

pages = {2776-2783},

abstract = {It is well-known that with Orbitrap-based Fourier-transform-mass-spectrometry (FT-MS) analysis, longer-time-domain signals are needed to better resolve species of interest. Unfortunately, increasing the signal-acquisition period comes at the expense of increasing ion decay, which lowers signal-to-noise ratios and ultimately limits resolution. This is especially problematic for intact proteins, including antibodies, which demonstrate rapid decay because of their larger collisional cross-sections, and result in more frequent collisions with background gas molecules. Provided here is a method that utilizes numerous low-ion-count spectra and single-ion processing to reconstruct a conventional m/ z spectrum. This technique has been applied to proteins varying in molecular weight from 8 to 150 kDa, with a resolving power of 677 000 achieved for transients of carbonic anhydrase (29 kDa) with a duration of only ∼250 ms. A resolution improvement ranging from 10- to 20-fold was observed for all proteins, providing isotopic resolution where none was previously present.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Swaroop2019,

title = {An activating mutation of the NSD2 histone methyltransferase drives oncogenic reprogramming in acute lymphocytic leukemia},

author = {Swaroop, A. and Oyer, J.A and Will, C.M and Huang, X and Yu, W and Troche, C and Bulic, M and Durham, B.H and Wen, Q.J and Crispino, J.D and MacKerell, A.D. Jr and Bennett, R.L and Kelleher, N.L and Licht, J.D. },

url = {https://www.nature.com/articles/s41388-018-0474-y},

doi = {10.1038/s41388-018-0474-y},

year  = {2019},

date = {2019-01-01},

journal = {Oncogene.},

volume = {38},

number = {5},

pages = {671-686},

abstract = {NSD2, a histone methyltransferase specific for methylation of histone 3 lysine 36 (H3K36), exhibits a glutamic acid to lysine mutation at residue 1099 (E1099K) in childhood acute lymphocytic leukemia (ALL), and cells harboring this mutation can become the predominant clone in relapsing disease. We studied the effects of this mutant enzyme in silico, in vitro, and in vivo using gene edited cell lines. The E1099K mutation altered enzyme/substrate binding and enhanced the rate of H3K36 methylation. As a result, cell lines harboring E1099K exhibit increased H3K36 dimethylation and reduced H3K27 trimethylation, particularly on nucleosomes containing histone H3.1. Mutant NSD2 cells exhibit reduced apoptosis and enhanced proliferation, clonogenicity, adhesion, and migration. In mouse xenografts, mutant NSD2 cells are more lethal and brain invasive than wildtype cells. Transcriptional profiling demonstrates that mutant NSD2 aberrantly activates factors commonly associated with neural and stromal lineages in addition to signaling and adhesion genes. Identification of these pathways provides new avenues for therapeutic interventions in NSD2 dysregulated malignancies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Jin2019,

title = {USP7 Cooperates with NOTCH1 to Drive the Oncogenic Transcriptional Program in T-Cell Leukemia},

author = {Jin, Q and Martinez, C.A and Arcipowski, K.M and Zhu, Y and Gutierrez-Diaz, B.T and Wang, K.K and Johnson, M.R and Volk, A.G and Wang, F and Wu, J and Grove, C and Wang, H and Sokirniy, I and Thomas, P.M and Goo, Y.A and Abshiru, N.A and Hijiya, N and Peirs, S and Vandamme, N and Berx, G and Goosens, S and Marshall, S.A and Rendleman, E.J and Takahashi, Y.H and Wang, L and Rawat, R and Bartom, E.T and Collings, C.K and Van Vlierberghe, P and Strikoudis, A and Kelly, S and Ueberheide, B and Mantis, C and Kandela, I and Bourquin, J.P and Bornhauser, B and Serafin, V and Bresolin, S and Paganin, M and Accordi, B and Basso, G and Kelleher, N.L and Weinstock, J and Kumar, S and Crispino, J.D and Shilatifard, A and Ntziachristos, P. },

url = {http://clincancerres.aacrjournals.org/content/25/1/222.long},

doi = {10.1158/1078-0432.CCR-18-1740},

year  = {2019},

date = {2019-01-01},

journal = {Clin Cancer Res.},

volume = {25},

number = {1},

pages = {222-239},

abstract = {T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes.



EXPERIMENTAL DESIGN:

To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models.



RESULTS:

We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo.



CONCLUSIONS:

These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Toby2019,

title = {A comprehensive pipeline for translational top-down proteomics from a single blood draw},

author = {Toby, T.K and Fornelli, L and Srzentić, K and DeHart, C.J and Levitsky, J and Friedewald, J and Kelleher, N.L.},

url = {https://www.nature.com/articles/s41596-018-0085-7},

doi = {10.1038/s41596-018-0085-7},

year  = {2019},

date = {2019-01-01},

journal = {Nat Protoc.},

volume = {14},

number = {1},

pages = {119-152},

abstract = {Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. However, the technique has been difficult to implement widely because of inherent limitations of existing sample preparation methods and instrumentation. Recent improvements in proteoform pre-fractionation and the availability of high-resolution benchtop mass spectrometers have made it possible to use high-throughput TDP for the analysis of complex clinical samples. Here, we provide a comprehensive protocol for analysis of a common sample type in translational research: human peripheral blood mononuclear cells (PBMCs). The pipeline comprises multiple workflows that can be treated as modular by the reader and used for various applications. First, sample collection and cell preservation are described for two clinical biorepository storage schemes. Cell lysis and proteoform pre-fractionation by gel-eluted liquid fractionation entrapment electrophoresis are then described. Importantly, instrument setup and liquid chromatography-tandem MS are described for TDP analyses, which rely on high-resolution Fourier-transform MS. Finally, data processing and analysis are described using two different, application-dependent software tools: ProSight Lite for targeted analyses of one or a few proteoforms and TDPortal for high-throughput TDP in discovery mode. For a single sample, the minimum completion time of the entire experiment is 72 h.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Alvares2018,

title = {The unique biomineralization transcriptome and proteome of Lytechinus variegatus teeth},

author = {Alvares, K and DeHart, C.J and Thomas, P.M and Kelleher, N.L and Veis, A. },

url = {https://www.tandfonline.com/doi/full/10.1080/03008207.2017.1408605},

doi = {10.1080/03008207.2017.1408605},

year  = {2018},

date = {2018-12-01},

journal = {Connect Tissue Res.},

volume = {59},

number = {sup1},

pages = {20-29},

abstract = {BACKGROUND:

Matrix-regulated biomineralization involves the specific nucleation and growth of mineral phases within or upon preformed structured organic matrices. We hypothesized that there might be a general mechanism whereby anionic, phosphorylated mineral ion-binding proteins assist in specifically locating the mineral ions with respect to the mineralizing structural organic matrix. Here we extended these studies to invertebrate mineralization in Lytechinus variegatus (Lv) teeth.



MATERIALS AND METHODS:

The tooth proteins were extracted and the phosphoproteins occluded in the mineral were enriched by passage through a ProQ Diamond phosphoprotein enrichment column, and subjected to MS/MS analysis. A Lv RNA-seq derived transcriptome database was generated. The MS/MS data found 25 proteins previously classified as "Predicted uncharacterized proteins" and many of the spicule matrix proteins. As these 25 proteins were also identified with the transcriptome analysis, and were thus no longer "hypothetical" but real proteins in the Lv tooth. Each protein was analyzed for the presence of a signal peptide, an acidic pI≤4, and the ability to be phosphorylated.



RESULTS:

Four new Lv tooth specific Pro-Ala-rich proteins were found, representing a new class of proteins.



CONCLUSION:

The tooth is different from the spicules and other urchin skeletal elements in that only the tooth contains both "high" and "very high" magnesium calcite, [Ca(1-X) Mg(X) CO3], where X is the mole fraction of Mg. We speculate that our newly discovered proline-alanine rich proteins, also containing sequences of acidic amino acids, may be involved in the formation of high magnesium and very high magnesium calcite.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Fisher2018,

title = {Characterization of a long overlooked copper protein from methane- and ammonia-oxidizing bacteria},

author = {Fisher, O.S and Kenney, G.E and Ross, M.O and Ro, S.Y and Lemma, B.E and Batelu, S and Thomas, P.M and Sosnowski, V.C and DeHart, C.J and Kelleher, N.L and Stemmler, T.L and Hoffman, B.M and Rosenzweig, A.C. },

url = {https://www.nature.com/articles/s41467-018-06681-5},

doi = {10.1038/s41467-018-06681-5},

year  = {2018},

date = {2018-10-15},

journal = {Nat Commun.},

volume = {9},

number = {1},

pages = {4276},

abstract = {Methane-oxidizing microbes catalyze the oxidation of the greenhouse gas methane using the copper-dependent enzyme particulate methane monooxygenase (pMMO). Isolated pMMO exhibits lower activity than whole cells, however, suggesting that additional components may be required. A pMMO homolog, ammonia monooxygenase (AMO), converts ammonia to hydroxylamine in ammonia-oxidizing bacteria (AOB) which produce another potent greenhouse gas, nitrous oxide. Here we show that PmoD, a protein encoded within many pmo operons that is homologous to the AmoD proteins encoded within AOB amo operons, forms a copper center that exhibits the features of a well-defined CuA site using a previously unobserved ligand set derived from a cupredoxin homodimer. PmoD is critical for copper-dependent growth on methane, and genetic analyses strongly support a role directly related to pMMO and AMO. These findings identify a copper-binding protein that may represent a missing link in the function of enzymes critical to the global carbon and nitrogen cycles.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mulowney2018,

title = {Natural products from thioester reductase containing biosynthetic pathways},

author = {Mullowney, M.W and McClure, R.A and Robey, M.T and Kelleher, N.L and Thomson, R.J. },

url = {https://pubs.rsc.org/en/content/articlelanding/2018/NP/C8NP00013A#!divAbstract},

doi = {10.1039/c8np00013a},

year  = {2018},

date = {2018-09-19},

journal = {Nat Prod Rep.},

volume = {35},

number = {9},

pages = {847-878},

abstract = {Covering: up to 2018 Thioester reductase domains catalyze two- and four-electron reductions to release natural products following assembly on nonribosomal peptide synthetases, polyketide synthases, and their hybrid biosynthetic complexes. This reductive off-loading of a natural product yields an aldehyde or alcohol, can initiate the formation of a macrocyclic imine, and contributes to important intermediates in a variety of biosyntheses, including those for polyketide alkaloids and pyrrolobenzodiazepines. Compounds that arise from reductase-terminated biosynthetic gene clusters are often reactive and exhibit biological activity. Biomedically important examples include the cancer therapeutic Yondelis (ecteinascidin 743), peptide aldehydes that inspired the first therapeutic proteasome inhibitor bortezomib, and numerous synthetic derivatives and antibody drug conjugates of the pyrrolobenzodiazepines. Recent advances in microbial genomics, metabolomics, bioinformatics, and reactivity-based labeling have facilitated the detection of these compounds for targeted isolation. Herein, we summarize known natural products arising from this important category, highlighting their occurrence in Nature, biosyntheses, biological activities, and the technologies used for their detection and identification. Additionally, we review publicly available genomic data to highlight the remaining potential for novel reductively tailored compounds and drug leads from microorganisms. This thorough retrospective highlights various molecular families with especially privileged bioactivity while illuminating challenges and prospects toward accelerating the discovery of new, high value natural products.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Gruppuso2018,

title = {Stability of histone post-translational modifications in samples derived from liver tissue and primary hepatic cells},

author = {Gruppuso, P.A and Boylan, J.M and Zabala, V and Neretti, N and Abshiru, N.A and Sikora, J.W and Doud, E.H. and Camarillo, J.M and Thomas, P.M and Kelleher, N.L and Sanders, J.A.. },

url = {https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0203351},

doi = {10.1371/journal.pone.0203351},

year  = {2018},

date = {2018-09-07},

journal = {PLoS One.},

volume = {13},

number = {9},

abstract = {Chromatin structure, a key contributor to the regulation of gene expression, is modulated by a broad array of histone post-translational modifications (PTMs). Taken together, these "histone marks" comprise what is often referred to as the "histone code". The quantitative analysis of histone PTMs by mass spectrometry (MS) offers the ability to examine the response of the histone code to physiological signals. However, few studies have examined the stability of histone PTMs through the process of isolating and culturing primary cells. To address this, we used bottom-up, MS-based analysis of histone PTMs in liver, freshly isolated hepatocytes, and cultured hepatocytes from adult male Fisher F344 rats. Correlations between liver, freshly isolated cells, and primary cultures were generally high, with R2 values exceeding 0.9. However, a number of acetylation marks, including those on H2A K9, H2A1 K13, H3 K4, H3 K14, H4 K8, H4 K12 and H4 K16 differed significantly among the three sources. Inducing proliferation of primary adult hepatocytes in culture affected several marks on histones H3.1/3.2 and H4. We conclude that hepatocyte isolation, culturing and cell cycle status all contribute to steady-state changes in the levels of a number of histone PTMs, indicating changes in histone marks that are rapidly induced in response to alterations in the cellular milieu. This has implications for studies aimed at assigning biological significance to histone modifications in tumors versus cancer cells, the developmental behavior of stem cells, and the attribution of changes in histone PTMs to altered cell metabolism.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Compton2018,

title = {Estimating the Distribution of Protein Post-Translational Modification States by Mass Spectrometry},

author = {Compton, P.D and Kelleher, N.L and Gunawardena, J. },

url = {https://pubs.acs.org/doi/10.1021/acs.jproteome.8b00150},

doi = {10.1021/acs.jproteome.8b00150},

year  = {2018},

date = {2018-08-03},

journal = {J Proteome Res.},

volume = {17},

number = {8},

pages = {2727-2734},

abstract = {Post-translational modifications (PTMs) of proteins play a central role in cellular information encoding, but the complexity of PTM state has been challenging to unravel. A single molecule can exhibit a "modform" or combinatorial pattern of co-occurring PTMs across multiple sites, and a molecular population can exhibit a distribution of amounts of different modforms. How can this "modform distribution" be estimated by mass spectrometry (MS)? Bottom-up MS, based on cleavage into peptides, destroys correlations between PTMs on different peptides, but it is conceivable that multiple proteases with appropriate patterns of cleavage could reconstruct the modform distribution. We introduce a mathematical language for describing MS measurements and show, on the contrary, that no matter how many distinct proteases are available, the shortfall in information required for reconstruction worsens exponentially with increasing numbers of sites. Whereas top-down MS on intact proteins can do better, current technology cannot prevent the exponential worsening. However, our analysis also shows that all forms of MS yield linear equations for modform amounts. This permits different MS protocols to be integrated and the modform distribution to be constrained within a high-dimensional "modform region", which may offer a feasible proxy for analyzing information encoding.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Fornelli2018,

title = {Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques},

author = {Fornelli, L and Srzentić, K and Huguet, R and Mullen, C and Sharma, S and Zabrouskov, V and Fellers, R.T and Durbin, K.R and Compton, P.D and Kelleher, N.L. },

url = {https://pubs.acs.org/doi/10.1021/acs.analchem.8b00984},

doi = {10.1021/acs.analchem.8b00984},

year  = {2018},

date = {2018-07-17},

journal = {Anal Chem.},

volume = {90},

number = {14},

pages = {8421-8429},

abstract = {Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Riley2018,

title = {The Value of Activated Ion Electron Transfer Dissociation for High-Throughput Top-Down Characterization of Intact Proteins},

author = {Riley, N.M and Sikora, J.W and Seckler, H.S and Greer, J.B and Fellers, R.T and LeDuc, R.D and Westphall, M.S and Thomas, P.M and Kelleher, N.L and Coon, J.J. },

url = {https://pubs.acs.org/doi/10.1021/acs.analchem.8b01638},

doi = {10.1021/acs.analchem.8b01638},

year  = {2018},

date = {2018-07-17},

journal = {Anal Chem.},

volume = {90},

number = {14},

pages = {8553-8560},

abstract = {High-throughput top-down proteomic experiments directly identify proteoforms in complex mixtures, making high quality tandem mass spectra necessary to deeply characterize proteins with many sources of variation. Collision-based dissociation methods offer expedient data acquisition but often fail to extensively fragment proteoforms for thorough analysis. Electron-driven dissociation methods are a popular alternative approach, especially for precursor ions with high charge density. Combining infrared photoactivation concurrent with electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), can significantly improve ETD characterization of intact proteins, but benefits of AI-ETD have yet to be quantified in high-throughput top-down proteomics. Here, we report the first application of AI-ETD to LC-MS/MS characterization of intact proteins (<20 kDa), highlighting improved proteoform identification the method offers over higher energy-collisional dissociation (HCD), standard ETD, and ETD followed by supplemental HCD activation (EThcD). We identified 935 proteoforms from 295 proteins from human colorectal cancer cell line HCT116 using AI-ETD compared to 1014 proteoforms, 915 proteoforms, and 871 proteoforms with HCD, ETD, and EThcD, respectively. Importantly, AI-ETD outperformed each of the three other methods in MS/MS success rates and spectral quality metrics (e.g., sequence coverage achieved and proteoform characterization scores). In all, this four-method analysis offers the most extensive comparisons to date and demonstrates that AI-ETD both increases identifications over other ETD methods and improves proteoform characterization via higher sequence coverage, positioning it as a premier method for high-throughput top-down proteomics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Park2018,

title = {Repurposed HisC Aminotransferases Complete the Biosynthesis of Some Methanobactins},

author = {Park, Y.J and Kenney, G.E and Schachner, L.F and Kelleher, N.L and Rosenzweig, A.C. },

url = {https://pubs.acs.org/doi/10.1021/acs.biochem.8b00296},

doi = {10.1021/acs.biochem.8b00296},

year  = {2018},

date = {2018-06-26},

journal = {Biochemistry},

volume = {57},

number = {25},

pages = {3515-3523},

abstract = {Methanobactins (Mbns) are ribosomally produced, post-translationally modified bacterial natural products with a high affinity for copper. MbnN, a pyridoxal 5'-phosphate-dependent aminotransferase, performs a transamination reaction that is the last step in the biosynthesis of Mbns produced by several Methylosinus species. Our bioinformatic analyses indicate that MbnNs likely derive from histidinol-phosphate aminotransferases (HisCs), which play a key role in histidine biosynthesis. A comparison of the HisC active site with the predicted MbnN structure suggests that MbnN's active site is altered to accommodate the larger and more hydrophobic substrates necessary for Mbn biosynthesis. Moreover, we have confirmed that MbnN is capable of catalyzing the final transamination step in Mbn biosynthesis in vitro and in vivo. We also demonstrate that without this final modification, Mbn exhibits significantly decreased stability under physiological conditions. An examination of other Mbns and Mbn operons suggests that N-terminal protection of this family of natural products is of critical importance and that several different means of N-terminal stabilization have evolved independently in Mbn subfamilies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Clevenger2018,

title = {Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity.},

author = {Clevenger, K.D. and Ye, R and Bok, J.W. and Thomas, P.M. and Islam, M.N. and Miley, G.P. and Robey, M.T. and Chen, C and Yang, K and Swyers, M and Wu, E and Gao, P and Wu, C.C. and Keller, N.P. and Kelleher, N.L.},

url = {https://pubs.acs.org/doi/10.1021/acs.biochem.8b00076},

doi = {10.1021/acs.biochem.8b00076},

year  = {2018},

date = {2018-06-12},

journal = {Biochemistry},

volume = {57},

number = {23},

pages = {3237-3243},

abstract = {The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Seckler2018,

title = {A Targeted, Differential Top-Down Proteomic Methodology for Comparison of ApoA-I Proteoforms in Individuals with High and Low HDL Efflux Capacity.},

author = {Seckler, H.D.S. and Fornelli, L and Mutharasan, R.K. and Thaxton, C.S. and Fellers, R and Daviglus, M and Sniderman, A and Rader, D and Kelleher, N.L. and Lloyd-Jones, D.M. and Compton, P.D. and Wilkins, J.T.},

url = {https://pubs.acs.org/doi/10.1021/acs.jproteome.8b00100},

doi = {10.1021/acs.jproteome.8b00100},

year  = {2018},

date = {2018-06-01},

journal = {J Proteome Res.},

volume = {17},

number = {6},

pages = {2156-2164},

abstract = {Top-down proteomics (TDP) allows precise determination/characterization of the different proteoforms derived from the expression of a single gene. In this study, we targeted apolipoprotein A-I (ApoA-I), a mediator of high-density-lipoprotein cholesterol efflux (HDL-E), which is inversely associated with coronary heart disease risk. Absolute ApoA-I concentration and allelic variation only partially explain interindividual HDL-E variation. Therefore, we hypothesize that differences in HDL-E are associated with the abundances of different ApoA-I proteoforms. Here, we present a targeted TDP methodology to characterize ApoA-I proteoforms in serum samples and compare their abundances between individuals. We characterized 18 ApoA-I proteoforms using selected-ion monitoring coupled to electron-transfer dissociation mass spectrometry. We then compared the abundances of these proteoforms between two groups of four participants, representing the individuals with highest and lowest HDL-E values within the Chicago Healthy Aging Study ( n = 420). Six proteoforms showed significantly ( p < 0.0005) higher intensity in high HDL-E individuals: canonical ApoA-I [fold difference (fd) = 1.17], carboxymethylated ApoA-I (fd = 1.24) and, with highest difference, four fatty acylated forms: palmitoylated (fd = 2.16), oleoylated (fd = 2.08), arachidonoylated (fd = 2.31) and one bearing two modifications: palmitoylation and truncation (fd = 2.13). These results demonstrate translational potential for targeted TDP in revealing, with high sensitivity, associations between interindividual proteoform variation and physiological differences underlying disease risk.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Robey2018,

title = {Identification of the First Diketomorpholine Biosynthetic Pathway Using FAC-MS Technology.},

author = {Robey, M.T. and Ye, R and Bok, J.W. and Clevenger, K.D. and Islam, M.N. and Chen, C and Gupta, R and Swyers, M and Wu, E and Gao, P and Thomas, P.M. and Wu, C.C. and Keller, N.P. and Kelleher, N.L.},

url = {https://pubs.acs.org/doi/10.1021/acschembio.8b00024},

doi = {10.1021/acschembio.8b00024},

year  = {2018},

date = {2018-05-18},

journal = {ACS Chem Biol.},

volume = {13},

number = {5},

pages = {1142-1147},

abstract = {Filamentous fungi are prolific producers of secondary metabolites with drug-like properties, and their genome sequences have revealed an untapped wealth of potential therapeutic leads. To better access these secondary metabolites and characterize their biosynthetic gene clusters, we applied a new platform for screening and heterologous expression of intact gene clusters that uses fungal artificial chromosomes and metabolomic scoring (FAC-MS). We leverage FAC-MS technology to identify the biosynthetic machinery responsible for production of acu-dioxomorpholine, a metabolite produced by the fungus, Aspergilllus aculeatus. The acu-dioxomorpholine nonribosomal peptide synthetase features a new type of condensation domain (designated CR) proposed to use a noncanonical arginine active site for ester bond formation. Using stable isotope labeling and MS, we determine that a phenyllactate monomer deriving from phenylalanine is incorporated into the diketomorpholine scaffold. Acu-dioxomorpholine is highly related to orphan inhibitors of P-glycoprotein targets in multidrug-resistant cancers, and identification of the biosynthetic pathway for this compound class enables genome mining for additional derivatives.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zheng2018,

title = {β-Glucocerebrosidase Modulators Promote Dimerization of β-Glucocerebrosidase and Reveal an Allosteric Binding Site},

author = {Zheng, J and Chen, L and Skinner, O.S and Ysselstein, D and Remis, J and Lansbury, P and Skerlj, R and Mrosek, M and Heunisch, U and Krapp, S and Charrow, J and Schwake, M and Kelleher, N.L and Silverman, R.B and Krainc, D},

url = {https://pubs.acs.org/doi/10.1021/jacs.7b13003},

doi = {10.1021/jacs.7b13003},

year  = {2018},

date = {2018-05-09},

journal = {J Am Chem Soc.},

volume = {140},

number = {18},

pages = {5914-5924},

abstract = {β-Glucocerebrosidase (GCase) mutations cause Gaucher's disease and are a high risk factor in Parkinson's disease. The implementation of a small molecule modulator is a strategy to restore proper folding and lysosome delivery of degradation-prone mutant GCase. Here, we present a potent quinazoline modulator, JZ-4109, which stabilizes wild-type and N370S mutant GCase and increases GCase abundance in patient-derived fibroblast cells. We then developed a covalent modification strategy using a lysine targeted inactivator (JZ-5029) for in vitro mechanistic studies. By using native top-down mass spectrometry, we located two potentially covalently modified lysines. We obtained the first crystal structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator covalently bound, and were able to identify the exact lysine residue modified (Lys346) and reveal an allosteric binding site. GCase dimerization was induced by our modulator binding, which was observed by native mass spectrometry, its crystal structure, and size exclusion chromatography with a multiangle light scattering detector. Finally, the dimer form was confirmed by negative staining transmission electron microscopy studies. Our newly discovered allosteric site and observed GCase dimerization provide a new mechanistic insight into GCase and its noniminosugar modulators and facilitate the rational design of novel GCase modulators for Gaucher's disease and Parkinson's disease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Parkinson2018,

title = {Discovery of the Tyrobetaine Natural Products and Their Biosynthetic Gene Cluster via Metabologenomics.},

author = {Parkinson, E.I. and Tryon, J.H. and Goering, A.W. and Ju, K.S. and McClure, R.A. and Kemball, J.D. and Zhukovsky, S and Labeda, D.P. and Thomson, R.J. and Kelleher, N.L. and Metcalf, W.W.},

url = {https://pubs.acs.org/doi/10.1021/acschembio.7b01089},

doi = {10.1021/acschembio.7b01089},

year  = {2018},

date = {2018-04-20},

journal = {ACS Chem Biol.},

volume = {13},

number = {4},

pages = {1029-1037},

abstract = {Natural products (NPs) are a rich source of medicines, but traditional discovery methods are often unsuccessful due to high rates of rediscovery. Genetic approaches for NP discovery are promising, but progress has been slow due to the difficulty of identifying unique biosynthetic gene clusters (BGCs) and poor gene expression. We previously developed the metabologenomics method, which combines genomic and metabolomic data to discover new NPs and their BGCs. Here, we utilize metabologenomics in combination with molecular networking to discover a novel class of NPs, the tyrobetaines: nonribosomal peptides with an unusual trimethylammonium tyrosine residue. The BGC for this unusual class of compounds was identified using metabologenomics and computational structure prediction data. Heterologous expression confirmed the BGC and suggests an unusual mechanism for trimethylammonium formation. Overall, the discovery of the tyrobetaines shows the great potential of metabologenomics combined with molecular networking and computational structure prediction for identifying interesting biosynthetic reactions and novel NPs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Miley2018,

title = {Total Synthesis of Tambromycin Enabled by Indole C-H Functionalization.},

author = {Miley, G.P. and Rote, J.C. and Silverman, R.B. and Kelleher, N.L. and Thomson, R.J.},

url = {https://pubs.acs.org/doi/10.1021/acs.orglett.8b00700},

doi = {10.1021/acs.orglett.8b00700},

year  = {2018},

date = {2018-04-20},

journal = {Org Lett.},

volume = {20},

number = {8},

pages = {2369-2373},

abstract = {The total synthesis of tambromycin (1), a recently isolated tetrapeptide, is reported. This unusual natural product possesses a highly modified tryptophan-derived indole fragment fused to an α-methylserine-derived oxazoline ring, and a unique noncanonical amino acid residue named tambroline (11). A convergent synthesis of tambromycin was achieved by a 13-step route that leveraged recent developments in the field of C-H functionalization to prepare the complex indole fragment, as well as an efficient synthesis of tambroline that featured a diastereoselective amination of homoproline.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ntai2018,

title = {Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk.},

author = {Ntai, I and Fornelli, L and DeHart, C.J. and Hutton, J.E. and Doubleday, P.F. and LeDuc, R.D. and van Nispen, A.J. and Fellers, R.T. and Whiteley, G and Boja, E.S. and Rodriguez, H and Kelleher, N.L.},

url = {https://www.pnas.org/content/115/16/4140.long},

doi = {10.1073/pnas.1716122115},

year  = {2018},

date = {2018-04-17},

journal = {Proc Natl Acad Sci USA},

volume = {115},

number = {16},

pages = {4140-4145},

abstract = {Mutations of the KRAS gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic KRAS colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kenney2018,

title = {The biosynthesis of methanobactin.},

author = {Kenney, G.E. and Dassama, L.M.K. and Pandelia, M.E. and Gizzi, A.S. and Martinie, R.J. and Gao, P and DeHart, C.J. and Schachner, L.F. and Skinner, O.S. and Ro, S.Y. and Zhu, X and Sadek, M and Thomas, P.M. and Almo, S.C. and Bollinger, J.M. and Jr, Krebs, C and Kelleher, N.L. and Rosenzweig, AC},

url = {https://science.sciencemag.org/content/359/6382/1411.long},

doi = {10.1126/science.aap9437},

year  = {2018},

date = {2018-03-23},

journal = {Science},

volume = {359},

number = {6382},

pages = {1411-1416},

abstract = {Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Martin2018,

title = {Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids.},

author = {Martin, R.W. and Des Soye, B.J. and Kwon, Y.C. and Kay, J and Davis, R.G. and Thomas, P.M. and Majewska, N.I. and Chen, C.X. and Marcum, R.D. and Weiss, M.G. and Stoddart, A.E. and Amiram, M and Ranji Charna, A.K. and Patel, J.R. and Isaacs, F.J. and Kelleher, N.L. and Hong, S.H. and Jewett, MC},

url = {https://www.nature.com/articles/s41467-018-03469-5},

doi = {10.1038/s41467-018-03469-5},

year  = {2018},

date = {2018-03-23},

journal = {Nat. Commun.},

volume = {9},

number = {1},

pages = {1203},

abstract = {Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Davis2018,

title = {Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains.},

author = {Davis, R.G. and Park, H.M. and Kim, K and Greer, J.B. and Fellers, R.T. and LeDuc, R.D. and Romanova, E.V. and Rubakhin, S.S. and Zombeck, J.A. and Wu, C and Yau, P.M. and Gao, P and van Nispen, A.J. and Patrie, S.M. and Thomas, P.M. and Sweedler, J.V. and Rhodes, J.S. and Kelleher, N.L.},

url = {https://pubs.acs.org/doi/10.1021/acs.analchem.7b04108},

doi = {10.1021/acs.analchem.7b04108},

year  = {2018},

date = {2018-03-20},

journal = {Anal Chem},

volume = {90},

number = {6},

pages = {3802-3810},

abstract = {Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Smith2018,

title = {Proteoforms as the next proteomics currency.},

author = {Smith, L.M. and Kelleher, N.L.},

url = {https://science.sciencemag.org/content/359/6380/1106.long},

doi = {10.1126/science.aat1884},

year  = {2018},

date = {2018-03-09},

journal = {Science},

volume = {359},

number = {6380},

pages = {1106-1107},

abstract = {Proteoforms—the different forms of proteins produced from the genome with a variety of sequence variations, splice isoforms, and myriad posttranslational modifications (1)—are critical elements in all biological systems (see the figure, left). Yang et al. (2) recently showed that the functions of proteins produced from splice variants from a given gene—different proteoforms—can be as different as those for proteins encoded by entirely different genes. Li et al. (3) showed that splice variants play a central role in modulating complex traits. However, the standard paradigm of proteomic analysis, the “bottom-up” strategy pioneered by Eng and Yates some 20 years ago (4), does not directly identify proteoforms. We argue that proteomic analysis needs to provide the identities and abundances of the proteoforms themselves, rather than just their peptide surrogates. Developing new proteome-wide strategies to accomplish this goal presents a formidable but not insurmountable technological challenge that will benefit the biomedical community.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{LeDuc2018,

title = {ProForma: A Standard Proteoform Notation.},

author = {LeDuc, R and Schwammle, V and Shortreed, MR and Cesnick, AJ and Solntsev, SK and Shaw, JB and Martin, MJ and Vizcaino, JA and Alpi,E and Danis, P and Kelleher, NL and Smith, LM and Ge, Y and Agar, JN and Chamot-Rooke, J and Loo, JA and Pasa-Tolic, L and Tsybin, YO},

url = {https://pubs.acs.org/doi/10.1021/acs.jproteome.7b00851},

doi = {10.1021/acs.jproteome.7b00851},

year  = {2018},

date = {2018-03-02},

journal = {J Proteome Res.},

volume = {17},

number = {3},

pages = {1321-1325},

abstract = {The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human-readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Aebersold2018,

title = {How many human proteoforms are there?},

author = {Aebersold, R and Agar, JN and Amster, IJ and Baker, MS and Bertozzi, CR and Boja, ES and Costello, CE and Cravatt, BF and Fenselau, C and Garcia, BA and Ge, Y and Gunawardena, J and Hendrickson, RC and Hergenrother, PJ and Huber, CG and Ivanov, AR and Jensen, ON and Jewett, MC and Kelleher, NL and Kiessling, LL and Krogan, NJ and Larsen, MR and Loo, JA and Ogorzalek, Loo RR and Lundberg, E and MacCoss, MJ and Mallick, P and Mootha, VK and Mrksich, M and Muir, TW and Patrie, SM and Pesavento, JJ and Pitteri, SJ and Rodriguez, H and Saghatelian, A and Sandoval, W and Schlüter, H and Sechi, S and Slavoff, SA and Smith, LM and Snyder, MP and Thomas, PM and Uhlén, M and Van Eyk, JE and Vidal, M and Walt, DR and White, FM and Williams, ER and Wohlschlager, T and Wysocki, VH and Yates, NA and Young, NL and Zhang, B},

url = {https://www.nature.com/articles/nchembio.2576},

doi = {10.1038/nchembio.2576},

year  = {2018},

date = {2018-02-14},

journal = {Nat Chem Biol.},

volume = {14},

number = {3},

pages = {206-214},

abstract = {Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Harris2017,

title = {The Mechanism of N2 Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H2},

author = {D. Harris and D. Lukoyanov and S. Shaw and P.D. Compton and M. Tokmina-Lukaszewska and B. Bothner and N.L. Kelleher and D.R. Dean and B.M. Hoffman and L.C. Seefeldt},

url = {http://pubs.acs.org/doi/10.1021/acs.biochem.7b01142},

doi = {10.1021/acs.biochem.7b01142},

year  = {2017},

date = {2017-12-28},

journal = {Biochemistry},

volume = {57},

number = {5},

pages = {701-710},

abstract = {Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), the Fe-nitrogenase has the poorest ratio of N2 reduction relative to H2 evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe-H-Fe hydrides on the active site FeMo-cofactor to yield H2 is a key feature in the N2 reduction mechanism. The N2 reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown. Here, we have purified both component proteins of the Fe-nitrogenase system, the electron-delivery Fe protein (AnfH) plus the catalytic FeFe protein (AnfDGK), and established its mechanism of N2 reduction. Inductively coupled plasma emission spectroscopy and protein mass spectrometry show that the FeFe protein component does not contain significant amounts of Mo or V, thus ruling out a requirement of these metals for N2 reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N2 reduction (1 atm) of 181 ± 5 nmol NH3 min-1 mg-1 FeFe protein, for proton reduction (in the absence of N2) of 1085 ± 41 nmol H2 min-1 mg-1 FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C2H4 min-1 mg-1 FeFe protein. Under turnover conditions N2 reduction is inhibited by H2 and the enzyme catalyzes the formation of HD when presented with N2 and D2. These observations are explained by the accumulation of four reducing equivalents as two metal-bound hydrides and two protons at the FeFe-cofactor, with activation for N2 reduction occurring by reductive elimination of H2.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Rickels2017,

title = {Histone H3K4 monomethylation catalyzed by Trr and mammalian COMPASS-like proteins at enhancers is dispensable for development and viability.},

author = {Rickels, R and Herz, H.M. and Sze, C.C. and Cao, K and Morgan, M.A. and Collings, C.K. and Gause, M and Takahashi, Y.H. and Wang, L and Rendleman, E.J. and Marshall, S.A. and Krueger, A and Bartom, E.T. and Piunti, A and Smith, E.R. and Abshiru, N.A. and Kelleher, N.L. and Dorsett, D and Shilatifard, A},

url = {https://www.nature.com/articles/ng.3965},

doi = {10.1038/ng.3965},

year  = {2017},

date = {2017-11-30},

journal = {Nat. Genet.},

volume = {49},

number = {11},

pages = {1647-1653},

abstract = {Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in Drosophila melanogaster and MLL3 (encoded by KMT2C) and MLL4 (encoded by KMT2D) in mammals. Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a trr allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal. Drosophila embryos with trr alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on cis-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{S.Turcan2017,

title = {Mutant-IDH1-dependent chromatin state reprogramming, reversibility, and persistence.},

author = {Turcan, S and Makarov, V and Taranda, J and Wang, Y and Fabius, A.W.M. and Wu, W and Zheng, Y and El-Amine, N and Haddock, S and Nanjangud, G and LeKaye, H.C. and Brennan, C and Cross, J and Huse, J.T. and Kelleher, N.L. and Osten, P and Thompson, C.B. and Chan, T.A.},

url = {https://www.nature.com/articles/s41588-017-0001-z},

doi = {10.1038/s41588-017-0001-z},

year  = {2017},

date = {2017-11-27},

journal = {Nat. Genet.},

volume = {50},

number = {1},

pages = {62-72},

abstract = {Mutations in IDH1 and IDH2 (encoding isocitrate dehydrogenase 1 and 2) drive the development of gliomas and other human malignancies. Mutant IDH1 induces epigenetic changes that promote tumorigenesis, but the scale and reversibility of these changes are unknown. Here, using human astrocyte and glioma tumorsphere systems, we generate a large-scale atlas of mutant-IDH1-induced epigenomic reprogramming. We characterize the reversibility of the alterations in DNA methylation, the histone landscape, and transcriptional reprogramming that occur following IDH1 mutation. We discover genome-wide coordinate changes in the localization and intensity of multiple histone marks and chromatin states. Mutant IDH1 establishes a CD24+ population with a proliferative advantage and stem-like transcriptional features. Strikingly, prolonged exposure to mutant IDH1 results in irreversible genomic and epigenetic alterations. Together, these observations provide unprecedented high-resolution molecular portraits of mutant-IDH1-dependent epigenomic reprogramming. These findings have substantial implications for understanding of mutant IDH function and for optimizing therapeutic approaches to targeting IDH-mutant tumors.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Skinner2017,

title = {Top-down characterization of endogenous protein complexes with native proteomics.},

author = {O.S. Skinner and N.A. Haverland and L. Fornelli and R.D. Melani and L.H.F. Do Vale and H.S. Seckler and P.F. Doubleday and L.F. Schachner and K. Srzentić and N.L. Kelleher and P.D. Compton},

url = {https://www.nature.com/articles/nchembio.2515},

doi = {10.1038/nchembio.2515},

year  = {2017},

date = {2017-11-13},

journal = {Nat. Chem. Biol.},

volume = {14},

number = {1},

pages = {36-41},

abstract = {Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein-protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mascarenhas2017b,

title = {Correction to Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover.},

author = {R. Mascarenhas and H.V. Le and K.D. Clevenger and H.J. Lehrer and D. Ringe and N.L. Kelleher and R.B. Silverman and D. Liu},

url = {http://pubs.acs.org/doi/10.1021/acs.biochem.7b00961},

doi = {10.1021/acs.biochem.7b00961},

year  = {2017},

date = {2017-10-31},

journal = {Biochemistry},

volume = {56},

number = {43},

pages = {5844-5845},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Skinner2017b,

title = {Native Electron Capture Dissociation Maps to Iron-Binding Channels in Horse Spleen Ferritin},

author = {O.S. Skinner and M.O. McAnally and R.P. Van Duyne and G.C. Schatz and K. Breuker and P.D. Compton and N.L. Kelleher},

url = {http://pubs.acs.org/doi/10.1021/acs.analchem.7b01581},

doi = {10.1021/acs.analchem.7b01581},

year  = {2017},

date = {2017-10-17},

journal = {Anal. Chem.},

volume = {89},

number = {20},

pages = {10711-10716},

abstract = {Native electron capture dissociation (NECD) is a process during which proteins undergo fragmentation similar to that from radical dissociation methods, but without the addition of exogenous electrons. However, after three initial reports of NECD from the cytochrome c dimer complex, no further evidence of the effect has been published. Here, we report NECD behavior from horse spleen ferritin, a ∼490 kDa protein complex ∼20-fold larger than the previously studied cytochrome c dimer. Application of front-end infrared excitation (FIRE) in conjunction with low- and high-m/z quadrupole isolation and collisionally activated dissociation (CAD) provides new insights into the NECD mechanism. Additionally, activation of the intact complex in either the electrospray droplet or the gas phase produced c-type fragment ions. Similar to the previously reported results on cytochrome c, these fragment ions form near residues known to interact with iron atoms in solution. By mapping the location of backbone cleavages associated with c-type ions onto the crystal structure, we are able to characterize two distinct iron binding channels that facilitate iron ion transport into the core of the complex. The resulting pathways are in good agreement with previously reported results for iron binding sites in mammalian ferritin.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Haverland2017,

title = {Cell Surface Proteomics of N-Linked Glycoproteins for Typing of Human Lymphocytes},

author = {N.A. Haverland and M. Wass and I. Ntai and T. Keppel and R.L. Gundry and N.L. Kelleher},

url = {http://onlinelibrary.wiley.com/doi/10.1002/pmic.201700156/epdf},

doi = {10.1002/pmic.201700156},

year  = {2017},

date = {2017-10-17},

journal = {Proteomics},

volume = {17},

number = {19},

abstract = {Lymphocytes are immune cells that are critical for the maintenance of adaptive immunity. Differentiation of lymphoid progenitors yields B-, T-, and NK-cell subtypes that individually correlate with specific forms of leukemia or lymphoma. Therefore, it is imperative a precise method of cell categorization is utilized to detect differences in distinct disease states present in patients. One viable means of classification involves evaluation of the cell surface proteome of lymphoid malignancies. Specifically, this manuscript details the use of an antibody independent approach known as Cell Surface Capture Technology, to assess the N-glycoproteome of four human lymphocyte cell lines. Altogether, 404 cell surface N-glycoproteins were identified as markers for specific cell types involved in lymphocytic malignancies, including 82 N-glycoproteins that had not been previously been described for B or T cells within the Cell Surface Protein Atlas. Comparative analysis, hierarchical clustering techniques, and label-free quantitation were used to reveal proteins most informative for each cell type. Undoubtedly, the characterization of the cell surface proteome of lymphoid malignancies is a first step toward improving personalized diagnosis and treatment of leukemia and lymphoma.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ezponda2017,

title = {UTX/KDM6A Loss Enhances the Malignant Phenotype of Multiple Myeloma and Sensitizes Cells to EZH2 inhibition},

author = {T. Ezponda and D. Dupéré-Richer and C.M. Will and E.C. Small and N. Varghese and T. Patel and B. Nabet and R. Popovic and J. Oyer and M. Bulic and Y. Zheng and X. Huang and M.Y. Shah and S. Maji and A. Riva and M. Occhionorelli and G. Tonon and N. Kelleher and J. Keats and J.D. Licht },

url = {http://www.sciencedirect.com/science/article/pii/S2211124717313839?via%3Dihub},

doi = {10.1016/j.celrep.2017.09.078},

year  = {2017},

date = {2017-10-17},

journal = {Cell. Rep.},

volume = {21},

number = {3},

pages = {628-640},

abstract = {Loss or inactivation of the histone H3K27 demethylase UTX occurs in several malignancies, including multiple myeloma (MM). Using an isogenic cell system, we found that loss of UTX leads to deactivation of gene expression ultimately promoting the proliferation, clonogenicity, adhesion, and tumorigenicity of MM cells. Moreover, UTX mutant cells showed increased in vitro and in vivo sensitivity to inhibition of EZH2, a histone methyltransferase that generates H3K27me3. Such sensitivity was related to a decrease in the levels of IRF4 and c-MYC and an activation of repressors of IRF4 characteristic of germinal center B cells such as BCL6 and IRF1. Rebalance of H3K27me3 levels at specific genes through EZH2 inhibitors may be a therapeutic strategy in MM cases harboring UTX mutations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zha2017,

title = {Colibactin assembly line enzymes use S-adenosylmethionine to build a cyclopropane ring.},

author = {L. Zha and Y. Jiang and M.T. Henke and M.R. Wilson and J.X. Wang and N.L. Kelleher and E.P. Balskus},

url = {http://dx.doi.org/10.1038/nchembio.2448},

doi = {10.1038/nchembio.2448},

year  = {2017},

date = {2017-10-01},

journal = {Nat Chem Biol},

volume = {13},

number = {10},

pages = {1063-1065},

abstract = {Despite containing an α-amino acid, the versatile cofactor S-adenosylmethionine (SAM) is not a known building block for nonribosomal peptide synthetase (NRPS) assembly lines. Here we report an unusual NRPS module from colibactin biosynthesis that uses SAM for amide bond formation and subsequent cyclopropanation. Our findings showcase a new use for SAM and reveal a novel biosynthetic route to a functional group that likely mediates colibactin's genotoxicity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Morgan2017,

title = {A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation.},

author = {M.A.J. Morgan and R.A. Rickels and C.K. Collings and X. He and K. Cao and H.M. Herz and K.A. Cozzolino and N.A. Abshiru and S.A. Marshall and E.J. Rendleman and C.C. Sze and A. Piunti and N.L. Kelleher and J.N. Savas and A. Shilatifard},

url = {http://genesdev.cshlp.org/content/31/19/2003.long},

doi = {10.1101/gad.305201.117.},

year  = {2017},

date = {2017-10-01},

journal = {Genes. Dev.},

volume = {19},

pages = {2003-2014},

abstract = {Histone H3 Lys4 (H3K4) methylation is a chromatin feature enriched at gene cis-regulatory sequences such as promoters and enhancers. Here we identify an evolutionarily conserved factor, BRWD2/PHIP, which colocalizes with histone H3K4 methylation genome-wide in human cells, mouse embryonic stem cells, and Drosophila Biochemical analysis of BRWD2 demonstrated an association with the Cullin-4-RING ubiquitin E3 ligase-4 (CRL4) complex, nucleosomes, and chromatin remodelers. BRWD2/PHIP binds directly to H3K4 methylation through a previously unidentified chromatin-binding module related to Royal Family Tudor domains, which we named the CryptoTudor domain. Using CRISPR-Cas9 genetic knockouts, we demonstrate that COMPASS H3K4 methyltransferase family members differentially regulate BRWD2/PHIP chromatin occupancy. Finally, we demonstrate that depletion of the single Drosophila homolog dBRWD3 results in altered gene expression and aberrant patterns of histone H3 Lys27 acetylation at enhancers and promoters, suggesting a cross-talk between these chromatin modifications and transcription through the BRWD protein family.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ichikawa2017,

title = {A synthetic biology approach to probing nucleosome symmetry},

author = {Y. Ichikawa and C.F. Connelly and A. Appleboim and T.C. Miller and H. Jacobi and N.A. Abshiru and H.J. Chou and Y. Chen and U. Sharma and Y. Zheng and P.M. Thomas and H.V. Chen and V. Bajaj and C.W. Müller and N.L. Kelleher and N. Friedman and D.N. Bolon and O.J. Rando and P.D. Kaufman},

url = {https://elifesciences.org/articles/28836},

doi = {10.7554/eLife.28836.},

year  = {2017},

date = {2017-09-12},

journal = {eLife},

number = {6},

abstract = {The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mascarenhas2017,

title = {Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover.},

author = {R. Mascarenhas and H.V. Le and K.D. Clevenger and H.J. Lehrer and D. Ringe and N.L. Kelleher and R.B. Silverman and D. Liu},

url = {https://dx.doi.org/10.1021/acs.biochem.7b00499},

doi = {10.1021/acs.biochem.7b00499},

year  = {2017},

date = {2017-09-06},

journal = {Biochemistry},

volume = {56},

number = {37},

pages = {4951-4961},

abstract = {Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Wildburger2017,

title = {Diversity of Amyloid-beta Proteoforms in the Alzheimer’s Disease Brain},

author = {N.C. Wildburger and T.J. Esparza and R.D. LeDuc and R.T. Fellers and P.M. Thomas and N.J. Cairns and N.L. Kelleher and R.J. Bateman and D.L. Brody},

url = {https://www.nature.com/articles/s41598-017-10422-x},

doi = {10.1038/s41598-017-10422-x},

year  = {2017},

date = {2017-08-25},

journal = {Sci. Rep.},

volume = {7},

number = {1},

pages = {9520},

abstract = {Amyloid-beta (Aβ) plays a key role in the pathogenesis of Alzheimer's disease (AD), but little is known about the proteoforms present in AD brain. We used high-resolution mass spectrometry to analyze intact Aβ from soluble aggregates and insoluble material in brains of six cases with severe dementia and pathologically confirmed AD. The soluble aggregates are especially relevant because they are believed to be the most toxic form of Aβ. We found a diversity of Aβ peptides, with 26 unique proteoforms including various N- and C-terminal truncations. N- and C-terminal truncations comprised 73% and 30%, respectively, of the total Aβ proteoforms detected. The Aβ proteoforms segregated between the soluble and more insoluble aggregates with N-terminal truncations predominating in the insoluble material and C- terminal truncations segregating into the soluble aggregates. In contrast, canonical Aβ comprised the minority of the identified proteoforms (15.3%) and did not distinguish between the soluble and more insoluble aggregates. The relative abundance of many truncated Aβ proteoforms did not correlate with post-mortem interval, suggesting they are not artefacts. This heterogeneity of Aβ proteoforms deepens our understanding of AD and offers many new avenues for investigation into pathological mechanisms of the disease, with implications for therapeutic development.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhou2017,

title = {Chromatographic efficiency and selectivity in top-down proteomics of histones.},

author = {Y. Zhou and X. Zhang and L. Fornelli and P.D. Compton and N. Kelleher and M.J. Wirth},

url = {http://www.sciencedirect.com/science/article/pii/S1570023216310972?via%3Dihub},

doi = {10.1016/j.jchromb.2016.12.027},

year  = {2017},

date = {2017-02-15},

journal = {J Chromatogr B Analyt Technol Biomed Life Sci},

pages = {47-53},

abstract = {Histones are involved in epigenetic control of a wide variety of cellular processes through their multiple post-translational modifications. Their strongly cationic nature makes them challenging to separate with reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS), where trifluoroacetic acid is avoided due to adduct formation. Columns with higher resolution are needed. In this work, RPLC-MS is performed on a histone sample using difluoroacetic acid and a 20-min gradient. Columns with C18 surfaces are compared for two different types of particle morphologies: 1) fully porous particles of 5μm in diameter, 2) superficially porous particles of 3μm in diameter with a shell of 0.2μm. The resolution for the histone separation is better for the latter column, but only when the modifier is trifluoroacetic acid, which is used with UV absorbance detection. When difluoroacetic acid is used for LCMS, the peaks broaden enough to erase the advantage in efficiency for the superficially porous particles. The fully porous and superficially porous cases show similar performance in RPLC-MS, with slightly higher resolution for the fully porous particles. The expected advantage of the shorter diffusion distances for the superficially porous particles is shown to be outweighed by the lower selectivity of its bonded phase.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN469,

title = {Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry},

author = { L. C. Anderson and C. J. DeHart and N. K. Kaiser and R. T. Fellers and D. F. Smith and J. B. Greer and R. D. LeDuc and G. T. Blakney and P. M. Thomas and N. L. Kelleher and C. L. Hendrickson},

url = {http://www.ncbi.nlm.nih.gov/pubmed/27936753},

doi = {10.1021/acs.jproteome.6b00696},

issn = {1535-3907 (Electronic) 

1535-3893 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {J Proteome Res},

volume = {16},

number = {2},

pages = {1087-1096},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN458,

title = {High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer},

author = { T. P. Cleland and C. J. DeHart and R. T. Fellers and A. J. VanNispen and J. B. Greer and R. D. LeDuc and W. R. Parker and P. M. Thomas and N. L. Kelleher and J. S. Brodbelt},

url = {http://www.ncbi.nlm.nih.gov/pubmed/28412815},

doi = {10.1021/acs.jproteome.7b00043},

issn = {1535-3907 (Electronic) 

1535-3893 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {J Proteome Res},

volume = {16},

number = {5},

pages = {2072-2079},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN456,

title = {A scalable platform to identify fungal secondary metabolites and their gene clusters},

author = { K. D. Clevenger and J. W. Bok and R. Ye and G. P. Miley and M. H. Verdan and T. Velk and C. Chen and K. Yang and M. T. Robey and P. Gao and M. Lamprecht and P. M. Thomas and M. N. Islam and J. M. Palmer and C. C. Wu and N. P. Keller and N. L. Kelleher},

url = {http://www.ncbi.nlm.nih.gov/pubmed/28604695},

doi = {10.1038/nchembio.2408},

issn = {1552-4469 (Electronic) 

1552-4450 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {Nat Chem Biol},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN464,

title = {Substrate Trapping in the Siderophore Tailoring Enzyme PvdQ},

author = { K. D. Clevenger and R. Mascarenhas and D. Catlin and R. Wu and N. L. Kelleher and E. J. Drake and A. M. Gulick and D. Liu and W. Fast},

url = {http://www.ncbi.nlm.nih.gov/pubmed/28186406},

doi = {10.1021/acschembio.7b00031},

issn = {1554-8937 (Electronic) 

1554-8929 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {ACS Chem Biol},

volume = {12},

number = {3},

pages = {643-647},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN466,

title = {Bioinformatics Analysis of Top-Down Mass Spectrometry Data with ProSight Lite},

author = { C. J. DeHart and R. T. Fellers and L. Fornelli and N. L. Kelleher and P. M. Thomas},

url = {http://www.ncbi.nlm.nih.gov/pubmed/28150248},

doi = {10.1007/978-1-4939-6783-4_18},

issn = {1940-6029 (Electronic) 

1064-3745 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {Methods Mol Biol},

volume = {1558},

pages = {381-394},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN465,

title = {Advancing Top-down Analysis of the Human Proteome Using a Benchtop Quadrupole-Orbitrap Mass Spectrometer},

author = { L. Fornelli and K. R. Durbin and R. T. Fellers and B. P. Early and J. B. Greer and R. D. LeDuc and P. D. Compton and N. L. Kelleher},

url = {http://www.ncbi.nlm.nih.gov/pubmed/28152595},

doi = {10.1021/acs.jproteome.6b00698},

issn = {1535-3907 (Electronic) 

1535-3893 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {J Proteome Res},

volume = {16},

number = {2},

pages = {609-618},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN463,

title = {Top-down proteomics: Where we are, where we are going?},

author = { L. Fornelli and T. K. Toby and L. F. Schachner and P. F. Doubleday and K. Srzentic and C. J. DeHart and N. L. Kelleher},

url = {http://www.ncbi.nlm.nih.gov/pubmed/28188863},

doi = {10.1016/j.jprot.2017.02.002},

issn = {1876-7737 (Electronic) 

1874-3919 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {J Proteomics},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN468,

title = {Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins},

author = { R. Gan and J. G. Perez and E. D. Carlson and I. Ntai and F. J. Isaacs and N. L. Kelleher and M. C. Jewett},

url = {http://www.ncbi.nlm.nih.gov/pubmed/27987323},

doi = {10.1002/bit.26239},

issn = {1097-0290 (Electronic) 

0006-3592 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {Biotechnol Bioeng},

volume = {114},

number = {5},

pages = {1074-1086},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN475,

title = {In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis},

author = { A. W. Goering and J. Li and R. A. McClure and R. J. Thomson and M. C. Jewett and N. L. Kelleher},

url = {http://www.ncbi.nlm.nih.gov/pubmed/27478992},

doi = {10.1021/acssynbio.6b00160},

issn = {2161-5063 (Electronic) 

2161-5063 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {ACS Synth Biol},

volume = {6},

number = {1},

pages = {39-44},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN459,

title = {Modulation of Protein Fragmentation Through Carbamylation of Primary Amines},

author = { S. M. Greer and D. D. Holden and R. Fellers and N. L. Kelleher and J. S. Brodbelt},

url = {http://www.ncbi.nlm.nih.gov/pubmed/28374316},

doi = {10.1007/s13361-017-1648-5},

issn = {1879-1123 (Electronic) 

1044-0305 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {J Am Soc Mass Spectrom},

volume = {28},

number = {8},

pages = {1587-1599},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN460,

title = {Defining Gas-Phase Fragmentation Propensities of Intact Proteins During Native Top-Down Mass Spectrometry},

author = { N. A. Haverland and O. S. Skinner and R. T. Fellers and A. A. Tariq and B. P. Early and R. D. LeDuc and L. Fornelli and P. D. Compton and N. L. Kelleher},

url = {http://www.ncbi.nlm.nih.gov/pubmed/28374312},

doi = {10.1007/s13361-017-1635-x},

issn = {1879-1123 (Electronic) 

1044-0305 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {J Am Soc Mass Spectrom},

volume = {28},

number = {6},

pages = {1203-1215},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN462,

title = {Characterizing the Structure and Oligomerization of Major Royal Jelly Protein 1 (MRJP1) by Mass Spectrometry and Complementary Biophysical Tools},

author = { S. C. Mandacaru and L. H. do Vale and S. Vahidi and Y. Xiao and O. S. Skinner and C. A. Ricart and N. L. Kelleher and M. V. de Sousa and L. Konermann},

url = {http://www.ncbi.nlm.nih.gov/pubmed/28252287},

doi = {10.1021/acs.biochem.7b00020},

issn = {1520-4995 (Electronic) 

0006-2960 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {Biochemistry},

volume = {56},

number = {11},

pages = {1645-1655},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN461,

title = {Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas},

author = { A. Piunti and R. Hashizume and M. A. Morgan and E. T. Bartom and C. M. Horbinski and S. A. Marshall and E. J. Rendleman and Q. Ma and Y. H. Takahashi and A. R. Woodfin and A. V. Misharin and N. A. Abshiru and R. R. Lulla and A. M. Saratsis and N. L. Kelleher and C. D. James and A. Shilatifard},

url = {http://www.ncbi.nlm.nih.gov/pubmed/28263307},

doi = {10.1038/nm.4296},

issn = {1546-170X (Electronic) 

1078-8956 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {Nat Med},

volume = {23},

number = {4},

pages = {493-500},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN467,

title = {Expansion for the Brachylophosaurus canadensis Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein},

author = { E. R. Schroeter and C. J. DeHart and T. P. Cleland and W. Zheng and P. M. Thomas and N. L. Kelleher and M. Bern and M. H. Schweitzer},

url = {http://www.ncbi.nlm.nih.gov/pubmed/28111950},

doi = {10.1021/acs.jproteome.6b00873},

issn = {1535-3907 (Electronic) 

1535-3893 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {J Proteome Res},

volume = {16},

number = {2},

pages = {920-932},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN457,

title = {Proteoforms in Peripheral Blood Mononuclear Cells as Novel Rejection Biomarkers in Liver Transplant Recipients},

author = { T. K. Toby and M. M. Abecassis and K. Kim and P. M. Thomas and R. T. Fellers and R. D. LeDuc and N. L. Kelleher and J. Demetris and J. Levitsky},

url = {http://www.ncbi.nlm.nih.gov/pubmed/28510335},

doi = {10.1111/ajt.14359},

issn = {1600-6143 (Electronic) 1600-6135 (Linking)},

year  = {2017},

date = {2017-01-01},

journal = {Am J Transplant},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN490,

title = {Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics},

author = { Y. Chen and R. A. McClure and N. L. Kelleher},

url = {http://www.ncbi.nlm.nih.gov/pubmed/26831706},

doi = {10.1007/978-1-4939-3375-4_9},

issn = {1940-6029 (Electronic) 

1064-3745 (Linking)},

year  = {2016},

date = {2016-01-01},

journal = {Methods Mol Biol},

volume = {1401},

pages = {135-47},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN491,

title = {Quantitation and Identification of Thousands of Human Proteoforms below 30 kDa},

author = { K. R. Durbin and L. Fornelli and R. T. Fellers and P. F. Doubleday and M. Narita and N. L. Kelleher},

url = {http://www.ncbi.nlm.nih.gov/pubmed/26795204},

doi = {10.1021/acs.jproteome.5b00997},

issn = {1535-3907 (Electronic) 

1535-3893 (Linking)},

year  = {2016},

date = {2016-01-01},

journal = {J Proteome Res},

volume = {15},

number = {3},

pages = {976-82},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN483,

title = {Metabologenomics: Correlation of Microbial Gene Clusters with Metabolites Drives Discovery of a Nonribosomal Peptide with an Unusual Amino Acid Monomer},

author = { A. W. Goering and R. A. McClure and J. R. Doroghazi and J. C. Albright and N. A. Haverland and Y. Zhang and K. S. Ju and R. J. Thomson and W. W. Metcalf and N. L. Kelleher},

url = {http://www.ncbi.nlm.nih.gov/pubmed/27163034},

doi = {10.1021/acscentsci.5b00331},

issn = {2374-7943 (Print) 

2374-7943 (Linking)},

year  = {2016},

date = {2016-01-01},

journal = {ACS Cent Sci},

volume = {2},

number = {2},

pages = {99-108},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN486,

title = {Enhanced binding of antibodies generated during chronic HIV infection to mucus component MUC16},

author = { B. Gunn and J. Schneider and M. Shansab and A. R. Bastian and K. Fahrbach and A. th Smith and A. Mahan and M. Karim and A. Licht and I. Zvonar and J. Tedesco and M. Anderson and A. Chapel and T. Suscovich and D. Malaspina and H. Streeck and B. D. Walker and A. Kim and G. Lauer and M. Altfeld and S. Pillai and I. Szleifer and N. L. Kelleher and P. F. Kiser and T. J. Hope and G. Alter},

url = {http://www.ncbi.nlm.nih.gov/pubmed/26960182},

doi = {10.1038/mi.2016.8},

issn = {1935-3456 (Electronic) 

1933-0219 (Linking)},

year  = {2016},

date = {2016-01-01},

journal = {Mucosal Immunol},

volume = {9},

number = {6},

pages = {1549-1558},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN489,

title = {Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation},

author = { T. Hattori and D. Lai and I. S. Dementieva and S. P. Montano and K. Kurosawa and Y. Zheng and L. R. Akin and K. M. Swist-Rosowska and A. T. Grzybowski and A. Koide and K. Krajewski and B. D. Strahl and N. L. Kelleher and A. J. Ruthenburg and S. Koide},

url = {http://www.ncbi.nlm.nih.gov/pubmed/26862167},

doi = {10.1073/pnas.1522691113},

issn = {1091-6490 (Electronic) 

0027-8424 (Linking)},

year  = {2016},

date = {2016-01-01},

journal = {Proc Natl Acad Sci U S A},

volume = {113},

number = {8},

pages = {2092-7},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN477,

title = {Modern mass spectrometry for synthetic biology and structure-based discovery of natural products},

author = { M. T. Henke and N. L. Kelleher},

url = {http://www.ncbi.nlm.nih.gov/pubmed/27376415},

doi = {10.1039/c6np00024j},

issn = {1460-4752 (Electronic) 

0265-0568 (Linking)},

year  = {2016},

date = {2016-01-01},

journal = {Nat Prod Rep},

volume = {33},

number = {8},

pages = {942-50},

keywords = {},

pubstate = {published},

tppubtype = {article}

}