Publications

@article{McGee2020b,

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},

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{Yusufova2020,

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},

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}

}