2020 |
Fornelli, L; Srzentić, K; Toby, T K; Doubleday, P F; Huguet, R; Mullen, C; Melani, R D; Dos Santos Seckler, H; DeHart, C J; Weisbrod, C R; Durbin, K R; Greer, J B; Early, B P; Fellers, R T; Zabrouskov, V; Thomas, P M; Compton, P D; Kelleher, N L Thorough Performance Evaluation of 213 nm Ultraviolet Photodissociation for Top-down Proteomics Journal Article Mol Cell Proteomics., 19 (2), pp. 405-420, 2020. Abstract | Links | BibTeX | Tags: @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} } 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. |
Kafader, J O; Melani, R D; Schachner, L F; Ives, A N; Patrie, S M; Kelleher, N L; Compton, P D Native vs Denatured: An in Depth Investigation of Charge State and Isotope Distributions Journal Article J Am Soc Mass Spectrom., 2020. Abstract | Links | BibTeX | Tags: @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.}, 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} } 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. |
Navarro-Muñoz, J C; Selem-Mojica, N; Mullowney, M W; Kautsar, S A; Tryon, J H; Parkinson, E I; De Los Santos, E L C; Yeong, M; Cruz-Morales, P; Abubucker, S; Roeters, A; Lokhorst, W; Fernandez-Guerra, A; Cappelini, L T D; Goering, A W; Thomson, R J; Metcalf, W W; Kelleher, N L; Barona-Gomez, F; Medema, M H A computational framework to explore large-scale biosynthetic diversity Journal Article Nat Chem Biol., 16 (1), pp. 60-68, 2020. Abstract | Links | BibTeX | Tags: @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} } 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. |
Park, H M; Winton, V J; Drader, J J; Manalili Wheeler, S; Lazar, G A; Kelleher, N L; Liu, Y; Tran, J C; Compton, P D Novel Interface for High-Throughput Analysis of Biotherapeutics by Electrospray Mass Spectrometry Journal Article Anal Chem., 92 (2), pp. 2186-2193, 2020. Abstract | Links | BibTeX | Tags: @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} } 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. |
Montaser, R; Kelleher, N L Discovery of the Biosynthetic Machinery for Stravidins, Biotin Antimetabolites Journal Article ACS Chem Biol. , 2020. Abstract | Links | BibTeX | Tags: @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} } 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. |
Winer, B; Edgel, K A; Zou, X; Sellau, J; Hadiwidjojo, S; Garver, L S; McDonough, C E; Kelleher, N L; Thomas, P M; Villasante, E; Ploss, A; Gerbasi, V R Identification of Plasmodium falciparum proteoforms from liver stage models Journal Article Malar J., 19 (1), pp. 10, 2020. Abstract | Links | BibTeX | Tags: @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} } 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. |
Doubleday, P F; Fornelli, L; Kelleher, N L Elucidating Proteoform Dynamics Underlying the Senescence Associated Secretory Phenotype Journal Article J Proteome Res., 2020. Abstract | Links | BibTeX | Tags: @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} } 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. |
2019 |
Camarillo, J M; Swaminathan, S; Abshiru, N A; Sikora, J W; Thomas, P M; Kelleher, N L Coupling Fluorescence-Activated Cell Sorting and Targeted Analysis of Histone Modification Profiles in Primary Human Leukocytes Journal Article J Am Soc Mass Spectrom., 30 (12), pp. 2526-2534, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Melani, R D; Srzentić, K; Gerbasi, V R; McGee, J P; Huguet, R; Fornelli, L; Kelleher, N L Direct measurement of light and heavy antibody chains using ion mobility and middle-down mass spectrometry Journal Article MAbs., 11 (8), pp. 1351-1357, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Des Soye, B J; Gerbasi, V R; Thomas, P M; Kelleher, N L; Jewett, M C A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli Journal Article Cell Chem Biol., 26 (12), pp. 1743-1754, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Huguet, R; Mullen, C; Srzentić, K; Greer, J B; Fellers, R T; Zabrouskov, V; Syka, J E P; Kelleher, N L; Fornelli, L Proton Transfer Charge Reduction Enables High-Throughput Top-Down Analysis of Large Proteoforms Journal Article Anal Chem., 91 (24), pp. 15732-15739, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Kafader, J O; Beu, S C; Early, B P; Melani, R D; Durbin, K R; Zabrouskov, V; Makarov, A A; Maze, J T; Shinholt, D L; Yip, P F; Kelleher, N L; Compton, P D; Senko, M W STORI Plots Enable Accurate Tracking of Individual Ion Signals Journal Article J Am Soc Mass Spectrom., 30 (11), pp. 2200-2203, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Park, H M; Satta, R; Davis, R G; Goo, Y A; LeDuc, R D; Fellers, R T; Greer, J B; Romanova, E V; Rubakhin, S S; Tai, R; Thomas, P M; Sweedler, J V; Kelleher, N L; Patrie, S M; Lasek, A W Multidimensional Top-Down Proteomics of Brain-Region-Specific Mouse Brain Proteoforms Responsive to Cocaine and Estradiol Journal Article J Proteome Res., 18 (11), pp. 3999-4012, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Schneider, J R; Shen, X; Orlandi, C; Nyanhete, T; Sawant, S; Carias, A M; Smith, A D; Kelleher, N L; Veazey, R S; Lewis, G K; Tomaras, G D; Hope, T J A MUC16 IgG binding activity selects for a restricted subset of IgG enriched for certain SIV epitope specificities Journal Article J Virol., 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Bennett, R L; Bele, A; Small, E C; Will, C M; Nabet, B; Oyer, J A; Huang, X; Ghosh, R P; Grzybowski, A T; Yu, T; Zhang, Q; Riva, A; Lele, T P; Schatz, G C; Kelleher, N L; Ruthenburg, A J; Liphardt, J; Licht, J D A Mutation in Histone H2B Represents a New Class of Oncogenic Driver Journal Article Cancer Discov., 9 (10), pp. 1438-1451, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Smith, L M; Thomas, P M; Shortreed, M R; Schaffer, L V; Fellers, R T; LeDuc, R D; Tucholski, T; Ge, Y; Agar, J N; Anderson, L C; Chamot-Rooke, J; Gault, J; Loo, J A; Paša-Tolić, L; Robinson, C V; Schlüter, H; Tsybin, Y O; Vilaseca, M; Vizcaíno, J A; Danis, P O; Kelleher, N L A five-level classification system for proteoform identifications Journal Article Nat Methods., 16 (10), pp. 939-940, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Lue, J K; Prabhu, S A; Liu, Y; Gonzalez, Y; Verma, A; Mundi, P S; Abshiru, N; Camarillo, J M; Mehta, S; Chen, E I; Qiao, C; Nandakumar, R; Cremers, S; Kelleher, N L; Elemento, O; Amengual, J E Precision Targeting with EZH2 and HDAC Inhibitors in Epigenetically Dysregulated Lymphomas Journal Article Clin Cancer Res, 25 (17), pp. 5271-5283, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Huang, X; LeDuc, R D; Fornelli, L; Schunter, A J; Bennett, R L; Kelleher, N L; Licht, J D Defining the NSD2 interactome: PARP1 PARylation reduces NSD2 histone methyltransferase activity and impedes chromatin binding Journal Article J Biol Chem., 294 (33), pp. 12459-12471, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Schachner, L F; Ives, A N; McGee, J P; Melani, R D; Kafader, J O; Compton, P D; Patrie, S M; Kelleher, N L Standard Proteoforms and Their Complexes for Native Mass Spectrometry Journal Article Journal of the American Society for Mass Spectrometry, 30 (7), pp. 1190-1198, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Donnelly, D P; Rawlins, C M; DeHart, C J; Fornelli, L; Schachner, L F; Lin, Z; Lippens, J L; Aluri, K C; Sarin, R; Chen, B; Lantz, C; Jung, W; Johnson, K R; Koller, A; Wolff, J J; Campuzano, I D G; Auclair, J R; Ivanov, A R; Whitelegge, J P; Paša-Tolić, L; Chamot-Rooke, J; Danis, P O; Smith, L M; Tsybin, Y O; Loo, J A; Ge, Y; Kelleher, N L; Agar, J N Best practices and benchmarks for intact protein analysis for top-down mass spectrometry Journal Article Nat Methods., 16 (7), pp. 587-594, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Moschitto, M J; Doubleday, P F; Catlin, D S; Kelleher, N L; Liu, D; Silverman, R B Mechanism of Inactivation of Ornithine Aminotransferase by (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)cyclopentane-1-carboxylic Acid Journal Article J Am Chem Soc., 14 (27), pp. 10711-10721, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Ro, S Y; Schachner, L F; Koo, C W; Purohit, R; Remis, J P; Kenney, G E; Liauw, B W; Thomas, P M; Patrie, S M; Kelleher, N L; Rosenzweig, A C Native top-down mass spectrometry provides insights into the copper centers of membrane-bound methane monooxygenase Journal Article Nat Commun., 10 (1), pp. 2675, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Schaffer, L V; Millikin, R J; Miller, R M; Anderson, L C; Fellers, R T; Ge, Y; Kelleher, N L; LeDuc, R D; Liu, X; Payne, S H; Sun, L; Thomas, P M; Tucholski, T; Wang, Z; Wu, S; Wu, Z; Yu, D; Shortreed, M R; Smith, L M Identification and Quantification of Proteoforms by Mass Spectrometry Journal Article Proteomics and Systems Biology, 19 (10), 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
LeDuc, R D; Fellers, R T; Early, B P; Greer, J B; Shams, D P; Thomas, P; Kelleher, N L Accurate Estimation of Context-Dependent False Discovery Rates in Top-Down Proteomics Journal Article Mol Cell Proteomics., 18 (4), pp. 796-805, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Cline, E N; Das, A; Bicca, M A; Mohammad, S N; Schachner, L F; Kamel, J M; DiNunno, N; Weng, A; Paschall, J D; Bu, R L; Khan, F M; Rollins, M G; Ives, A N; Shekhawat, G; Nunes-Tavares, N; de Mello, F G; Compton, P D; Kelleher, N L; Klein, W L A novel crosslinking protocol stabilizes amyloid β oligomers capable of inducing Alzheimer's-associated pathologies Journal Article J Neurochem., 148 (6), pp. 822-836, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Kafader, J O; Melani, R D; Senko, M W; Makarov, A A; Kelleher, N L; Compton, P D Measurement of Individual Ions Sharply Increases the Resolution of Orbitrap Mass Spectra of Proteins Journal Article Anal Chem., 91 (4), pp. 2776-2783, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Swaroop, A; Oyer, J A; Will, C M; Huang, X; Yu, W; Troche, C; Bulic, M; Durham, B H; Wen, Q J; Crispino, J D; MacKerell, Jr A D; Bennett, R L; Kelleher, N L; Licht, J D An activating mutation of the NSD2 histone methyltransferase drives oncogenic reprogramming in acute lymphocytic leukemia Journal Article Oncogene., 38 (5), pp. 671-686, 2019. Abstract | Links | BibTeX | Tags: @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} } 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. |
Jin, Q; Martinez, C A; Arcipowski, K M; Zhu, Y; Gutierrez-Diaz, B T; Wang, K K; Johnson, M R; Volk, A G; Wang, F; Wu, J; Grove, C; Wang, H; Sokirniy, I; Thomas, P M; Goo, Y A; Abshiru, N A; Hijiya, N; Peirs, S; Vandamme, N; Berx, G; Goosens, S; Marshall, S A; Rendleman, E J; Takahashi, Y H; Wang, L; Rawat, R; Bartom, E T; Collings, C K; Van Vlierberghe, P; Strikoudis, A; Kelly, S; Ueberheide, B; Mantis, C; Kandela, I; Bourquin, J P; Bornhauser, B; Serafin, V; Bresolin, S; Paganin, M; Accordi, B; Basso, G; Kelleher, N L; Weinstock, J; Kumar, S; Crispino, J D; Shilatifard, A; Ntziachristos, P USP7 Cooperates with NOTCH1 to Drive the Oncogenic Transcriptional Program in T-Cell Leukemia Journal Article Clin Cancer Res., 25 (1), pp. 222-239, 2019. Abstract | Links | BibTeX | Tags: @article{Jin2019, title = {USP7 Cooperates with NOTCH1 to Drive the Oncogenic Transcriptional Program in T-Cell Leukemia}, author = {Jin, Q and Martinez, C.A and Arcipowski, K.M and Zhu, Y and Gutierrez-Diaz, B.T and Wang, K.K and Johnson, M.R and Volk, A.G and Wang, F and Wu, J and Grove, C and Wang, H and Sokirniy, I and Thomas, P.M and Goo, Y.A and Abshiru, N.A and Hijiya, N and Peirs, S and Vandamme, N and Berx, G and Goosens, S and Marshall, S.A and Rendleman, E.J and Takahashi, Y.H and Wang, L and Rawat, R and Bartom, E.T and Collings, C.K and Van Vlierberghe, P and Strikoudis, A and Kelly, S and Ueberheide, B and Mantis, C and Kandela, I and Bourquin, J.P and Bornhauser, B and Serafin, V and Bresolin, S and Paganin, M and Accordi, B and Basso, G and Kelleher, N.L and Weinstock, J and Kumar, S and Crispino, J.D and Shilatifard, A and Ntziachristos, P. }, url = {http://clincancerres.aacrjournals.org/content/25/1/222.long}, doi = {10.1158/1078-0432.CCR-18-1740}, year = {2019}, date = {2019-01-01}, journal = {Clin Cancer Res.}, volume = {25}, number = {1}, pages = {222-239}, abstract = {T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. EXPERIMENTAL DESIGN: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. RESULTS: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. CONCLUSIONS: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.}, keywords = {}, pubstate = {published}, tppubtype = {article} } T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. EXPERIMENTAL DESIGN: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. RESULTS: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. CONCLUSIONS: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia. |
Toby, T K; Fornelli, L; Srzentić, K; DeHart, C J; Levitsky, J; Friedewald, J; Kelleher, N L A comprehensive pipeline for translational top-down proteomics from a single blood draw Journal Article Nat Protoc., 14 (1), pp. 119-152, 2019. Abstract | Links | BibTeX | Tags: @article{Toby2019, title = {A comprehensive pipeline for translational top-down proteomics from a single blood draw}, author = {Toby, T.K and Fornelli, L and Srzentić, K and DeHart, C.J and Levitsky, J and Friedewald, J and Kelleher, N.L.}, url = {https://www.nature.com/articles/s41596-018-0085-7}, doi = {10.1038/s41596-018-0085-7}, year = {2019}, date = {2019-01-01}, journal = {Nat Protoc.}, volume = {14}, number = {1}, pages = {119-152}, abstract = {Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. However, the technique has been difficult to implement widely because of inherent limitations of existing sample preparation methods and instrumentation. Recent improvements in proteoform pre-fractionation and the availability of high-resolution benchtop mass spectrometers have made it possible to use high-throughput TDP for the analysis of complex clinical samples. Here, we provide a comprehensive protocol for analysis of a common sample type in translational research: human peripheral blood mononuclear cells (PBMCs). The pipeline comprises multiple workflows that can be treated as modular by the reader and used for various applications. First, sample collection and cell preservation are described for two clinical biorepository storage schemes. Cell lysis and proteoform pre-fractionation by gel-eluted liquid fractionation entrapment electrophoresis are then described. Importantly, instrument setup and liquid chromatography-tandem MS are described for TDP analyses, which rely on high-resolution Fourier-transform MS. Finally, data processing and analysis are described using two different, application-dependent software tools: ProSight Lite for targeted analyses of one or a few proteoforms and TDPortal for high-throughput TDP in discovery mode. For a single sample, the minimum completion time of the entire experiment is 72 h.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. However, the technique has been difficult to implement widely because of inherent limitations of existing sample preparation methods and instrumentation. Recent improvements in proteoform pre-fractionation and the availability of high-resolution benchtop mass spectrometers have made it possible to use high-throughput TDP for the analysis of complex clinical samples. Here, we provide a comprehensive protocol for analysis of a common sample type in translational research: human peripheral blood mononuclear cells (PBMCs). The pipeline comprises multiple workflows that can be treated as modular by the reader and used for various applications. First, sample collection and cell preservation are described for two clinical biorepository storage schemes. Cell lysis and proteoform pre-fractionation by gel-eluted liquid fractionation entrapment electrophoresis are then described. Importantly, instrument setup and liquid chromatography-tandem MS are described for TDP analyses, which rely on high-resolution Fourier-transform MS. Finally, data processing and analysis are described using two different, application-dependent software tools: ProSight Lite for targeted analyses of one or a few proteoforms and TDPortal for high-throughput TDP in discovery mode. For a single sample, the minimum completion time of the entire experiment is 72 h. |
2018 |
Alvares, K; DeHart, C J; Thomas, P M; Kelleher, N L; Veis, A The unique biomineralization transcriptome and proteome of Lytechinus variegatus teeth Journal Article Connect Tissue Res., 59 (sup1), pp. 20-29, 2018. Abstract | Links | BibTeX | Tags: @article{Alvares2018, title = {The unique biomineralization transcriptome and proteome of Lytechinus variegatus teeth}, author = {Alvares, K and DeHart, C.J and Thomas, P.M and Kelleher, N.L and Veis, A. }, url = {https://www.tandfonline.com/doi/full/10.1080/03008207.2017.1408605}, doi = {10.1080/03008207.2017.1408605}, year = {2018}, date = {2018-12-01}, journal = {Connect Tissue Res.}, volume = {59}, number = {sup1}, pages = {20-29}, abstract = {BACKGROUND: Matrix-regulated biomineralization involves the specific nucleation and growth of mineral phases within or upon preformed structured organic matrices. We hypothesized that there might be a general mechanism whereby anionic, phosphorylated mineral ion-binding proteins assist in specifically locating the mineral ions with respect to the mineralizing structural organic matrix. Here we extended these studies to invertebrate mineralization in Lytechinus variegatus (Lv) teeth. MATERIALS AND METHODS: The tooth proteins were extracted and the phosphoproteins occluded in the mineral were enriched by passage through a ProQ Diamond phosphoprotein enrichment column, and subjected to MS/MS analysis. A Lv RNA-seq derived transcriptome database was generated. The MS/MS data found 25 proteins previously classified as "Predicted uncharacterized proteins" and many of the spicule matrix proteins. As these 25 proteins were also identified with the transcriptome analysis, and were thus no longer "hypothetical" but real proteins in the Lv tooth. Each protein was analyzed for the presence of a signal peptide, an acidic pI≤4, and the ability to be phosphorylated. RESULTS: Four new Lv tooth specific Pro-Ala-rich proteins were found, representing a new class of proteins. CONCLUSION: The tooth is different from the spicules and other urchin skeletal elements in that only the tooth contains both "high" and "very high" magnesium calcite, [Ca(1-X) Mg(X) CO3], where X is the mole fraction of Mg. We speculate that our newly discovered proline-alanine rich proteins, also containing sequences of acidic amino acids, may be involved in the formation of high magnesium and very high magnesium calcite.}, keywords = {}, pubstate = {published}, tppubtype = {article} } BACKGROUND: Matrix-regulated biomineralization involves the specific nucleation and growth of mineral phases within or upon preformed structured organic matrices. We hypothesized that there might be a general mechanism whereby anionic, phosphorylated mineral ion-binding proteins assist in specifically locating the mineral ions with respect to the mineralizing structural organic matrix. Here we extended these studies to invertebrate mineralization in Lytechinus variegatus (Lv) teeth. MATERIALS AND METHODS: The tooth proteins were extracted and the phosphoproteins occluded in the mineral were enriched by passage through a ProQ Diamond phosphoprotein enrichment column, and subjected to MS/MS analysis. A Lv RNA-seq derived transcriptome database was generated. The MS/MS data found 25 proteins previously classified as "Predicted uncharacterized proteins" and many of the spicule matrix proteins. As these 25 proteins were also identified with the transcriptome analysis, and were thus no longer "hypothetical" but real proteins in the Lv tooth. Each protein was analyzed for the presence of a signal peptide, an acidic pI≤4, and the ability to be phosphorylated. RESULTS: Four new Lv tooth specific Pro-Ala-rich proteins were found, representing a new class of proteins. CONCLUSION: The tooth is different from the spicules and other urchin skeletal elements in that only the tooth contains both "high" and "very high" magnesium calcite, [Ca(1-X) Mg(X) CO3], where X is the mole fraction of Mg. We speculate that our newly discovered proline-alanine rich proteins, also containing sequences of acidic amino acids, may be involved in the formation of high magnesium and very high magnesium calcite. |
Fisher, O S; Kenney, G E; Ross, M O; Ro, S Y; Lemma, B E; Batelu, S; Thomas, P M; Sosnowski, V C; DeHart, C J; Kelleher, N L; Stemmler, T L; Hoffman, B M; Rosenzweig, A C Characterization of a long overlooked copper protein from methane- and ammonia-oxidizing bacteria Journal Article Nat Commun., 9 (1), pp. 4276, 2018. Abstract | Links | BibTeX | Tags: @article{Fisher2018, title = {Characterization of a long overlooked copper protein from methane- and ammonia-oxidizing bacteria}, author = {Fisher, O.S and Kenney, G.E and Ross, M.O and Ro, S.Y and Lemma, B.E and Batelu, S and Thomas, P.M and Sosnowski, V.C and DeHart, C.J and Kelleher, N.L and Stemmler, T.L and Hoffman, B.M and Rosenzweig, A.C. }, url = {https://www.nature.com/articles/s41467-018-06681-5}, doi = {10.1038/s41467-018-06681-5}, year = {2018}, date = {2018-10-15}, journal = {Nat Commun.}, volume = {9}, number = {1}, pages = {4276}, abstract = {Methane-oxidizing microbes catalyze the oxidation of the greenhouse gas methane using the copper-dependent enzyme particulate methane monooxygenase (pMMO). Isolated pMMO exhibits lower activity than whole cells, however, suggesting that additional components may be required. A pMMO homolog, ammonia monooxygenase (AMO), converts ammonia to hydroxylamine in ammonia-oxidizing bacteria (AOB) which produce another potent greenhouse gas, nitrous oxide. Here we show that PmoD, a protein encoded within many pmo operons that is homologous to the AmoD proteins encoded within AOB amo operons, forms a copper center that exhibits the features of a well-defined CuA site using a previously unobserved ligand set derived from a cupredoxin homodimer. PmoD is critical for copper-dependent growth on methane, and genetic analyses strongly support a role directly related to pMMO and AMO. These findings identify a copper-binding protein that may represent a missing link in the function of enzymes critical to the global carbon and nitrogen cycles.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Methane-oxidizing microbes catalyze the oxidation of the greenhouse gas methane using the copper-dependent enzyme particulate methane monooxygenase (pMMO). Isolated pMMO exhibits lower activity than whole cells, however, suggesting that additional components may be required. A pMMO homolog, ammonia monooxygenase (AMO), converts ammonia to hydroxylamine in ammonia-oxidizing bacteria (AOB) which produce another potent greenhouse gas, nitrous oxide. Here we show that PmoD, a protein encoded within many pmo operons that is homologous to the AmoD proteins encoded within AOB amo operons, forms a copper center that exhibits the features of a well-defined CuA site using a previously unobserved ligand set derived from a cupredoxin homodimer. PmoD is critical for copper-dependent growth on methane, and genetic analyses strongly support a role directly related to pMMO and AMO. These findings identify a copper-binding protein that may represent a missing link in the function of enzymes critical to the global carbon and nitrogen cycles. |
Mullowney, M W; McClure, R A; Robey, M T; Kelleher, N L; Thomson, R J Natural products from thioester reductase containing biosynthetic pathways Journal Article Nat Prod Rep., 35 (9), pp. 847-878, 2018. Abstract | Links | BibTeX | Tags: @article{Mulowney2018, title = {Natural products from thioester reductase containing biosynthetic pathways}, author = {Mullowney, M.W and McClure, R.A and Robey, M.T and Kelleher, N.L and Thomson, R.J. }, url = {https://pubs.rsc.org/en/content/articlelanding/2018/NP/C8NP00013A#!divAbstract}, doi = {10.1039/c8np00013a}, year = {2018}, date = {2018-09-19}, journal = {Nat Prod Rep.}, volume = {35}, number = {9}, pages = {847-878}, abstract = {Covering: up to 2018 Thioester reductase domains catalyze two- and four-electron reductions to release natural products following assembly on nonribosomal peptide synthetases, polyketide synthases, and their hybrid biosynthetic complexes. This reductive off-loading of a natural product yields an aldehyde or alcohol, can initiate the formation of a macrocyclic imine, and contributes to important intermediates in a variety of biosyntheses, including those for polyketide alkaloids and pyrrolobenzodiazepines. Compounds that arise from reductase-terminated biosynthetic gene clusters are often reactive and exhibit biological activity. Biomedically important examples include the cancer therapeutic Yondelis (ecteinascidin 743), peptide aldehydes that inspired the first therapeutic proteasome inhibitor bortezomib, and numerous synthetic derivatives and antibody drug conjugates of the pyrrolobenzodiazepines. Recent advances in microbial genomics, metabolomics, bioinformatics, and reactivity-based labeling have facilitated the detection of these compounds for targeted isolation. Herein, we summarize known natural products arising from this important category, highlighting their occurrence in Nature, biosyntheses, biological activities, and the technologies used for their detection and identification. Additionally, we review publicly available genomic data to highlight the remaining potential for novel reductively tailored compounds and drug leads from microorganisms. This thorough retrospective highlights various molecular families with especially privileged bioactivity while illuminating challenges and prospects toward accelerating the discovery of new, high value natural products.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Covering: up to 2018 Thioester reductase domains catalyze two- and four-electron reductions to release natural products following assembly on nonribosomal peptide synthetases, polyketide synthases, and their hybrid biosynthetic complexes. This reductive off-loading of a natural product yields an aldehyde or alcohol, can initiate the formation of a macrocyclic imine, and contributes to important intermediates in a variety of biosyntheses, including those for polyketide alkaloids and pyrrolobenzodiazepines. Compounds that arise from reductase-terminated biosynthetic gene clusters are often reactive and exhibit biological activity. Biomedically important examples include the cancer therapeutic Yondelis (ecteinascidin 743), peptide aldehydes that inspired the first therapeutic proteasome inhibitor bortezomib, and numerous synthetic derivatives and antibody drug conjugates of the pyrrolobenzodiazepines. Recent advances in microbial genomics, metabolomics, bioinformatics, and reactivity-based labeling have facilitated the detection of these compounds for targeted isolation. Herein, we summarize known natural products arising from this important category, highlighting their occurrence in Nature, biosyntheses, biological activities, and the technologies used for their detection and identification. Additionally, we review publicly available genomic data to highlight the remaining potential for novel reductively tailored compounds and drug leads from microorganisms. This thorough retrospective highlights various molecular families with especially privileged bioactivity while illuminating challenges and prospects toward accelerating the discovery of new, high value natural products. |
Gruppuso, P A; Boylan, J M; Zabala, V; Neretti, N; Abshiru, N A; Sikora, J W; Doud, E H; Camarillo, J M; Thomas, P M; Kelleher, N L; Sanders, J A Stability of histone post-translational modifications in samples derived from liver tissue and primary hepatic cells Journal Article PLoS One., 13 (9), 2018. Abstract | Links | BibTeX | Tags: @article{Gruppuso2018, title = {Stability of histone post-translational modifications in samples derived from liver tissue and primary hepatic cells}, author = {Gruppuso, P.A and Boylan, J.M and Zabala, V and Neretti, N and Abshiru, N.A and Sikora, J.W and Doud, E.H. and Camarillo, J.M and Thomas, P.M and Kelleher, N.L and Sanders, J.A.. }, url = {https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0203351}, doi = {10.1371/journal.pone.0203351}, year = {2018}, date = {2018-09-07}, journal = {PLoS One.}, volume = {13}, number = {9}, abstract = {Chromatin structure, a key contributor to the regulation of gene expression, is modulated by a broad array of histone post-translational modifications (PTMs). Taken together, these "histone marks" comprise what is often referred to as the "histone code". The quantitative analysis of histone PTMs by mass spectrometry (MS) offers the ability to examine the response of the histone code to physiological signals. However, few studies have examined the stability of histone PTMs through the process of isolating and culturing primary cells. To address this, we used bottom-up, MS-based analysis of histone PTMs in liver, freshly isolated hepatocytes, and cultured hepatocytes from adult male Fisher F344 rats. Correlations between liver, freshly isolated cells, and primary cultures were generally high, with R2 values exceeding 0.9. However, a number of acetylation marks, including those on H2A K9, H2A1 K13, H3 K4, H3 K14, H4 K8, H4 K12 and H4 K16 differed significantly among the three sources. Inducing proliferation of primary adult hepatocytes in culture affected several marks on histones H3.1/3.2 and H4. We conclude that hepatocyte isolation, culturing and cell cycle status all contribute to steady-state changes in the levels of a number of histone PTMs, indicating changes in histone marks that are rapidly induced in response to alterations in the cellular milieu. This has implications for studies aimed at assigning biological significance to histone modifications in tumors versus cancer cells, the developmental behavior of stem cells, and the attribution of changes in histone PTMs to altered cell metabolism.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Chromatin structure, a key contributor to the regulation of gene expression, is modulated by a broad array of histone post-translational modifications (PTMs). Taken together, these "histone marks" comprise what is often referred to as the "histone code". The quantitative analysis of histone PTMs by mass spectrometry (MS) offers the ability to examine the response of the histone code to physiological signals. However, few studies have examined the stability of histone PTMs through the process of isolating and culturing primary cells. To address this, we used bottom-up, MS-based analysis of histone PTMs in liver, freshly isolated hepatocytes, and cultured hepatocytes from adult male Fisher F344 rats. Correlations between liver, freshly isolated cells, and primary cultures were generally high, with R2 values exceeding 0.9. However, a number of acetylation marks, including those on H2A K9, H2A1 K13, H3 K4, H3 K14, H4 K8, H4 K12 and H4 K16 differed significantly among the three sources. Inducing proliferation of primary adult hepatocytes in culture affected several marks on histones H3.1/3.2 and H4. We conclude that hepatocyte isolation, culturing and cell cycle status all contribute to steady-state changes in the levels of a number of histone PTMs, indicating changes in histone marks that are rapidly induced in response to alterations in the cellular milieu. This has implications for studies aimed at assigning biological significance to histone modifications in tumors versus cancer cells, the developmental behavior of stem cells, and the attribution of changes in histone PTMs to altered cell metabolism. |
Compton, P D; Kelleher, N L; Gunawardena, J Estimating the Distribution of Protein Post-Translational Modification States by Mass Spectrometry Journal Article J Proteome Res., 17 (8), pp. 2727-2734, 2018. Abstract | Links | BibTeX | Tags: @article{Compton2018, title = {Estimating the Distribution of Protein Post-Translational Modification States by Mass Spectrometry}, author = {Compton, P.D and Kelleher, N.L and Gunawardena, J. }, url = {https://pubs.acs.org/doi/10.1021/acs.jproteome.8b00150}, doi = {10.1021/acs.jproteome.8b00150}, year = {2018}, date = {2018-08-03}, journal = {J Proteome Res.}, volume = {17}, number = {8}, pages = {2727-2734}, abstract = {Post-translational modifications (PTMs) of proteins play a central role in cellular information encoding, but the complexity of PTM state has been challenging to unravel. A single molecule can exhibit a "modform" or combinatorial pattern of co-occurring PTMs across multiple sites, and a molecular population can exhibit a distribution of amounts of different modforms. How can this "modform distribution" be estimated by mass spectrometry (MS)? Bottom-up MS, based on cleavage into peptides, destroys correlations between PTMs on different peptides, but it is conceivable that multiple proteases with appropriate patterns of cleavage could reconstruct the modform distribution. We introduce a mathematical language for describing MS measurements and show, on the contrary, that no matter how many distinct proteases are available, the shortfall in information required for reconstruction worsens exponentially with increasing numbers of sites. Whereas top-down MS on intact proteins can do better, current technology cannot prevent the exponential worsening. However, our analysis also shows that all forms of MS yield linear equations for modform amounts. This permits different MS protocols to be integrated and the modform distribution to be constrained within a high-dimensional "modform region", which may offer a feasible proxy for analyzing information encoding.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Post-translational modifications (PTMs) of proteins play a central role in cellular information encoding, but the complexity of PTM state has been challenging to unravel. A single molecule can exhibit a "modform" or combinatorial pattern of co-occurring PTMs across multiple sites, and a molecular population can exhibit a distribution of amounts of different modforms. How can this "modform distribution" be estimated by mass spectrometry (MS)? Bottom-up MS, based on cleavage into peptides, destroys correlations between PTMs on different peptides, but it is conceivable that multiple proteases with appropriate patterns of cleavage could reconstruct the modform distribution. We introduce a mathematical language for describing MS measurements and show, on the contrary, that no matter how many distinct proteases are available, the shortfall in information required for reconstruction worsens exponentially with increasing numbers of sites. Whereas top-down MS on intact proteins can do better, current technology cannot prevent the exponential worsening. However, our analysis also shows that all forms of MS yield linear equations for modform amounts. This permits different MS protocols to be integrated and the modform distribution to be constrained within a high-dimensional "modform region", which may offer a feasible proxy for analyzing information encoding. |
Fornelli, L; Srzentić, K; Huguet, R; Mullen, C; Sharma, S; Zabrouskov, V; Fellers, R T; Durbin, K R; Compton, P D; Kelleher, N L Anal Chem., 90 (14), pp. 8421-8429, 2018. Abstract | Links | BibTeX | Tags: @article{Fornelli2018, title = {Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques}, author = {Fornelli, L and Srzentić, K and Huguet, R and Mullen, C and Sharma, S and Zabrouskov, V and Fellers, R.T and Durbin, K.R and Compton, P.D and Kelleher, N.L. }, url = {https://pubs.acs.org/doi/10.1021/acs.analchem.8b00984}, doi = {10.1021/acs.analchem.8b00984}, year = {2018}, date = {2018-07-17}, journal = {Anal Chem.}, volume = {90}, number = {14}, pages = {8421-8429}, abstract = {Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies. |
Riley, N M; Sikora, J W; Seckler, H S; Greer, J B; Fellers, R T; LeDuc, R D; Westphall, M S; Thomas, P M; Kelleher, N L; Coon, J J The Value of Activated Ion Electron Transfer Dissociation for High-Throughput Top-Down Characterization of Intact Proteins Journal Article Anal Chem., 90 (14), pp. 8553-8560, 2018. Abstract | Links | BibTeX | Tags: @article{Riley2018, title = {The Value of Activated Ion Electron Transfer Dissociation for High-Throughput Top-Down Characterization of Intact Proteins}, author = {Riley, N.M and Sikora, J.W and Seckler, H.S and Greer, J.B and Fellers, R.T and LeDuc, R.D and Westphall, M.S and Thomas, P.M and Kelleher, N.L and Coon, J.J. }, url = {https://pubs.acs.org/doi/10.1021/acs.analchem.8b01638}, doi = {10.1021/acs.analchem.8b01638}, year = {2018}, date = {2018-07-17}, journal = {Anal Chem.}, volume = {90}, number = {14}, pages = {8553-8560}, abstract = {High-throughput top-down proteomic experiments directly identify proteoforms in complex mixtures, making high quality tandem mass spectra necessary to deeply characterize proteins with many sources of variation. Collision-based dissociation methods offer expedient data acquisition but often fail to extensively fragment proteoforms for thorough analysis. Electron-driven dissociation methods are a popular alternative approach, especially for precursor ions with high charge density. Combining infrared photoactivation concurrent with electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), can significantly improve ETD characterization of intact proteins, but benefits of AI-ETD have yet to be quantified in high-throughput top-down proteomics. Here, we report the first application of AI-ETD to LC-MS/MS characterization of intact proteins (<20 kDa), highlighting improved proteoform identification the method offers over higher energy-collisional dissociation (HCD), standard ETD, and ETD followed by supplemental HCD activation (EThcD). We identified 935 proteoforms from 295 proteins from human colorectal cancer cell line HCT116 using AI-ETD compared to 1014 proteoforms, 915 proteoforms, and 871 proteoforms with HCD, ETD, and EThcD, respectively. Importantly, AI-ETD outperformed each of the three other methods in MS/MS success rates and spectral quality metrics (e.g., sequence coverage achieved and proteoform characterization scores). In all, this four-method analysis offers the most extensive comparisons to date and demonstrates that AI-ETD both increases identifications over other ETD methods and improves proteoform characterization via higher sequence coverage, positioning it as a premier method for high-throughput top-down proteomics.}, keywords = {}, pubstate = {published}, tppubtype = {article} } High-throughput top-down proteomic experiments directly identify proteoforms in complex mixtures, making high quality tandem mass spectra necessary to deeply characterize proteins with many sources of variation. Collision-based dissociation methods offer expedient data acquisition but often fail to extensively fragment proteoforms for thorough analysis. Electron-driven dissociation methods are a popular alternative approach, especially for precursor ions with high charge density. Combining infrared photoactivation concurrent with electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), can significantly improve ETD characterization of intact proteins, but benefits of AI-ETD have yet to be quantified in high-throughput top-down proteomics. Here, we report the first application of AI-ETD to LC-MS/MS characterization of intact proteins (<20 kDa), highlighting improved proteoform identification the method offers over higher energy-collisional dissociation (HCD), standard ETD, and ETD followed by supplemental HCD activation (EThcD). We identified 935 proteoforms from 295 proteins from human colorectal cancer cell line HCT116 using AI-ETD compared to 1014 proteoforms, 915 proteoforms, and 871 proteoforms with HCD, ETD, and EThcD, respectively. Importantly, AI-ETD outperformed each of the three other methods in MS/MS success rates and spectral quality metrics (e.g., sequence coverage achieved and proteoform characterization scores). In all, this four-method analysis offers the most extensive comparisons to date and demonstrates that AI-ETD both increases identifications over other ETD methods and improves proteoform characterization via higher sequence coverage, positioning it as a premier method for high-throughput top-down proteomics. |
Park, Y J; Kenney, G E; Schachner, L F; Kelleher, N L; Rosenzweig, A C Repurposed HisC Aminotransferases Complete the Biosynthesis of Some Methanobactins Journal Article Biochemistry, 57 (25), pp. 3515-3523, 2018. Abstract | Links | BibTeX | Tags: @article{Park2018, title = {Repurposed HisC Aminotransferases Complete the Biosynthesis of Some Methanobactins}, author = {Park, Y.J and Kenney, G.E and Schachner, L.F and Kelleher, N.L and Rosenzweig, A.C. }, url = {https://pubs.acs.org/doi/10.1021/acs.biochem.8b00296}, doi = {10.1021/acs.biochem.8b00296}, year = {2018}, date = {2018-06-26}, journal = {Biochemistry}, volume = {57}, number = {25}, pages = {3515-3523}, abstract = {Methanobactins (Mbns) are ribosomally produced, post-translationally modified bacterial natural products with a high affinity for copper. MbnN, a pyridoxal 5'-phosphate-dependent aminotransferase, performs a transamination reaction that is the last step in the biosynthesis of Mbns produced by several Methylosinus species. Our bioinformatic analyses indicate that MbnNs likely derive from histidinol-phosphate aminotransferases (HisCs), which play a key role in histidine biosynthesis. A comparison of the HisC active site with the predicted MbnN structure suggests that MbnN's active site is altered to accommodate the larger and more hydrophobic substrates necessary for Mbn biosynthesis. Moreover, we have confirmed that MbnN is capable of catalyzing the final transamination step in Mbn biosynthesis in vitro and in vivo. We also demonstrate that without this final modification, Mbn exhibits significantly decreased stability under physiological conditions. An examination of other Mbns and Mbn operons suggests that N-terminal protection of this family of natural products is of critical importance and that several different means of N-terminal stabilization have evolved independently in Mbn subfamilies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Methanobactins (Mbns) are ribosomally produced, post-translationally modified bacterial natural products with a high affinity for copper. MbnN, a pyridoxal 5'-phosphate-dependent aminotransferase, performs a transamination reaction that is the last step in the biosynthesis of Mbns produced by several Methylosinus species. Our bioinformatic analyses indicate that MbnNs likely derive from histidinol-phosphate aminotransferases (HisCs), which play a key role in histidine biosynthesis. A comparison of the HisC active site with the predicted MbnN structure suggests that MbnN's active site is altered to accommodate the larger and more hydrophobic substrates necessary for Mbn biosynthesis. Moreover, we have confirmed that MbnN is capable of catalyzing the final transamination step in Mbn biosynthesis in vitro and in vivo. We also demonstrate that without this final modification, Mbn exhibits significantly decreased stability under physiological conditions. An examination of other Mbns and Mbn operons suggests that N-terminal protection of this family of natural products is of critical importance and that several different means of N-terminal stabilization have evolved independently in Mbn subfamilies. |
Clevenger, K D; Ye, R; Bok, J W; Thomas, P M; Islam, M N; Miley, G P; Robey, M T; Chen, C; Yang, K; Swyers, M; Wu, E; Gao, P; Wu, C C; Keller, N P; Kelleher, N L Biochemistry, 57 (23), pp. 3237-3243, 2018. Abstract | Links | BibTeX | Tags: @article{Clevenger2018, title = {Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity.}, author = {Clevenger, K.D. and Ye, R and Bok, J.W. and Thomas, P.M. and Islam, M.N. and Miley, G.P. and Robey, M.T. and Chen, C and Yang, K and Swyers, M and Wu, E and Gao, P and Wu, C.C. and Keller, N.P. and Kelleher, N.L.}, url = {https://pubs.acs.org/doi/10.1021/acs.biochem.8b00076}, doi = {10.1021/acs.biochem.8b00076}, year = {2018}, date = {2018-06-12}, journal = {Biochemistry}, volume = {57}, number = {23}, pages = {3237-3243}, abstract = {The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities. |
Seckler, H D S; Fornelli, L; Mutharasan, R K; Thaxton, C S; Fellers, R; Daviglus, M; Sniderman, A; Rader, D; Kelleher, N L; Lloyd-Jones, D M; Compton, P D; Wilkins, J T J Proteome Res., 17 (6), pp. 2156-2164, 2018. Abstract | Links | BibTeX | Tags: @article{Seckler2018, title = {A Targeted, Differential Top-Down Proteomic Methodology for Comparison of ApoA-I Proteoforms in Individuals with High and Low HDL Efflux Capacity.}, author = {Seckler, H.D.S. and Fornelli, L and Mutharasan, R.K. and Thaxton, C.S. and Fellers, R and Daviglus, M and Sniderman, A and Rader, D and Kelleher, N.L. and Lloyd-Jones, D.M. and Compton, P.D. and Wilkins, J.T.}, url = {https://pubs.acs.org/doi/10.1021/acs.jproteome.8b00100}, doi = {10.1021/acs.jproteome.8b00100}, year = {2018}, date = {2018-06-01}, journal = {J Proteome Res.}, volume = {17}, number = {6}, pages = {2156-2164}, abstract = {Top-down proteomics (TDP) allows precise determination/characterization of the different proteoforms derived from the expression of a single gene. In this study, we targeted apolipoprotein A-I (ApoA-I), a mediator of high-density-lipoprotein cholesterol efflux (HDL-E), which is inversely associated with coronary heart disease risk. Absolute ApoA-I concentration and allelic variation only partially explain interindividual HDL-E variation. Therefore, we hypothesize that differences in HDL-E are associated with the abundances of different ApoA-I proteoforms. Here, we present a targeted TDP methodology to characterize ApoA-I proteoforms in serum samples and compare their abundances between individuals. We characterized 18 ApoA-I proteoforms using selected-ion monitoring coupled to electron-transfer dissociation mass spectrometry. We then compared the abundances of these proteoforms between two groups of four participants, representing the individuals with highest and lowest HDL-E values within the Chicago Healthy Aging Study ( n = 420). Six proteoforms showed significantly ( p < 0.0005) higher intensity in high HDL-E individuals: canonical ApoA-I [fold difference (fd) = 1.17], carboxymethylated ApoA-I (fd = 1.24) and, with highest difference, four fatty acylated forms: palmitoylated (fd = 2.16), oleoylated (fd = 2.08), arachidonoylated (fd = 2.31) and one bearing two modifications: palmitoylation and truncation (fd = 2.13). These results demonstrate translational potential for targeted TDP in revealing, with high sensitivity, associations between interindividual proteoform variation and physiological differences underlying disease risk.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Top-down proteomics (TDP) allows precise determination/characterization of the different proteoforms derived from the expression of a single gene. In this study, we targeted apolipoprotein A-I (ApoA-I), a mediator of high-density-lipoprotein cholesterol efflux (HDL-E), which is inversely associated with coronary heart disease risk. Absolute ApoA-I concentration and allelic variation only partially explain interindividual HDL-E variation. Therefore, we hypothesize that differences in HDL-E are associated with the abundances of different ApoA-I proteoforms. Here, we present a targeted TDP methodology to characterize ApoA-I proteoforms in serum samples and compare their abundances between individuals. We characterized 18 ApoA-I proteoforms using selected-ion monitoring coupled to electron-transfer dissociation mass spectrometry. We then compared the abundances of these proteoforms between two groups of four participants, representing the individuals with highest and lowest HDL-E values within the Chicago Healthy Aging Study ( n = 420). Six proteoforms showed significantly ( p < 0.0005) higher intensity in high HDL-E individuals: canonical ApoA-I [fold difference (fd) = 1.17], carboxymethylated ApoA-I (fd = 1.24) and, with highest difference, four fatty acylated forms: palmitoylated (fd = 2.16), oleoylated (fd = 2.08), arachidonoylated (fd = 2.31) and one bearing two modifications: palmitoylation and truncation (fd = 2.13). These results demonstrate translational potential for targeted TDP in revealing, with high sensitivity, associations between interindividual proteoform variation and physiological differences underlying disease risk. |
Robey, M T; Ye, R; Bok, J W; Clevenger, K D; Islam, M N; Chen, C; Gupta, R; Swyers, M; Wu, E; Gao, P; Thomas, P M; Wu, C C; Keller, N P; Kelleher, N L Identification of the First Diketomorpholine Biosynthetic Pathway Using FAC-MS Technology. Journal Article ACS Chem Biol., 13 (5), pp. 1142-1147, 2018. Abstract | Links | BibTeX | Tags: @article{Robey2018, title = {Identification of the First Diketomorpholine Biosynthetic Pathway Using FAC-MS Technology.}, author = {Robey, M.T. and Ye, R and Bok, J.W. and Clevenger, K.D. and Islam, M.N. and Chen, C and Gupta, R and Swyers, M and Wu, E and Gao, P and Thomas, P.M. and Wu, C.C. and Keller, N.P. and Kelleher, N.L.}, url = {https://pubs.acs.org/doi/10.1021/acschembio.8b00024}, doi = {10.1021/acschembio.8b00024}, year = {2018}, date = {2018-05-18}, journal = {ACS Chem Biol.}, volume = {13}, number = {5}, pages = {1142-1147}, abstract = {Filamentous fungi are prolific producers of secondary metabolites with drug-like properties, and their genome sequences have revealed an untapped wealth of potential therapeutic leads. To better access these secondary metabolites and characterize their biosynthetic gene clusters, we applied a new platform for screening and heterologous expression of intact gene clusters that uses fungal artificial chromosomes and metabolomic scoring (FAC-MS). We leverage FAC-MS technology to identify the biosynthetic machinery responsible for production of acu-dioxomorpholine, a metabolite produced by the fungus, Aspergilllus aculeatus. The acu-dioxomorpholine nonribosomal peptide synthetase features a new type of condensation domain (designated CR) proposed to use a noncanonical arginine active site for ester bond formation. Using stable isotope labeling and MS, we determine that a phenyllactate monomer deriving from phenylalanine is incorporated into the diketomorpholine scaffold. Acu-dioxomorpholine is highly related to orphan inhibitors of P-glycoprotein targets in multidrug-resistant cancers, and identification of the biosynthetic pathway for this compound class enables genome mining for additional derivatives.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Filamentous fungi are prolific producers of secondary metabolites with drug-like properties, and their genome sequences have revealed an untapped wealth of potential therapeutic leads. To better access these secondary metabolites and characterize their biosynthetic gene clusters, we applied a new platform for screening and heterologous expression of intact gene clusters that uses fungal artificial chromosomes and metabolomic scoring (FAC-MS). We leverage FAC-MS technology to identify the biosynthetic machinery responsible for production of acu-dioxomorpholine, a metabolite produced by the fungus, Aspergilllus aculeatus. The acu-dioxomorpholine nonribosomal peptide synthetase features a new type of condensation domain (designated CR) proposed to use a noncanonical arginine active site for ester bond formation. Using stable isotope labeling and MS, we determine that a phenyllactate monomer deriving from phenylalanine is incorporated into the diketomorpholine scaffold. Acu-dioxomorpholine is highly related to orphan inhibitors of P-glycoprotein targets in multidrug-resistant cancers, and identification of the biosynthetic pathway for this compound class enables genome mining for additional derivatives. |
Zheng, J; Chen, L; Skinner, O S; Ysselstein, D; Remis, J; Lansbury, P; Skerlj, R; Mrosek, M; Heunisch, U; Krapp, S; Charrow, J; Schwake, M; Kelleher, N L; Silverman, R B; Krainc, D β-Glucocerebrosidase Modulators Promote Dimerization of β-Glucocerebrosidase and Reveal an Allosteric Binding Site Journal Article J Am Chem Soc., 140 (18), pp. 5914-5924, 2018. Abstract | Links | BibTeX | Tags: @article{Zheng2018, title = {β-Glucocerebrosidase Modulators Promote Dimerization of β-Glucocerebrosidase and Reveal an Allosteric Binding Site}, author = {Zheng, J and Chen, L and Skinner, O.S and Ysselstein, D and Remis, J and Lansbury, P and Skerlj, R and Mrosek, M and Heunisch, U and Krapp, S and Charrow, J and Schwake, M and Kelleher, N.L and Silverman, R.B and Krainc, D}, url = {https://pubs.acs.org/doi/10.1021/jacs.7b13003}, doi = {10.1021/jacs.7b13003}, year = {2018}, date = {2018-05-09}, journal = {J Am Chem Soc.}, volume = {140}, number = {18}, pages = {5914-5924}, abstract = {β-Glucocerebrosidase (GCase) mutations cause Gaucher's disease and are a high risk factor in Parkinson's disease. The implementation of a small molecule modulator is a strategy to restore proper folding and lysosome delivery of degradation-prone mutant GCase. Here, we present a potent quinazoline modulator, JZ-4109, which stabilizes wild-type and N370S mutant GCase and increases GCase abundance in patient-derived fibroblast cells. We then developed a covalent modification strategy using a lysine targeted inactivator (JZ-5029) for in vitro mechanistic studies. By using native top-down mass spectrometry, we located two potentially covalently modified lysines. We obtained the first crystal structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator covalently bound, and were able to identify the exact lysine residue modified (Lys346) and reveal an allosteric binding site. GCase dimerization was induced by our modulator binding, which was observed by native mass spectrometry, its crystal structure, and size exclusion chromatography with a multiangle light scattering detector. Finally, the dimer form was confirmed by negative staining transmission electron microscopy studies. Our newly discovered allosteric site and observed GCase dimerization provide a new mechanistic insight into GCase and its noniminosugar modulators and facilitate the rational design of novel GCase modulators for Gaucher's disease and Parkinson's disease.}, keywords = {}, pubstate = {published}, tppubtype = {article} } β-Glucocerebrosidase (GCase) mutations cause Gaucher's disease and are a high risk factor in Parkinson's disease. The implementation of a small molecule modulator is a strategy to restore proper folding and lysosome delivery of degradation-prone mutant GCase. Here, we present a potent quinazoline modulator, JZ-4109, which stabilizes wild-type and N370S mutant GCase and increases GCase abundance in patient-derived fibroblast cells. We then developed a covalent modification strategy using a lysine targeted inactivator (JZ-5029) for in vitro mechanistic studies. By using native top-down mass spectrometry, we located two potentially covalently modified lysines. We obtained the first crystal structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator covalently bound, and were able to identify the exact lysine residue modified (Lys346) and reveal an allosteric binding site. GCase dimerization was induced by our modulator binding, which was observed by native mass spectrometry, its crystal structure, and size exclusion chromatography with a multiangle light scattering detector. Finally, the dimer form was confirmed by negative staining transmission electron microscopy studies. Our newly discovered allosteric site and observed GCase dimerization provide a new mechanistic insight into GCase and its noniminosugar modulators and facilitate the rational design of novel GCase modulators for Gaucher's disease and Parkinson's disease. |
Parkinson, E I; Tryon, J H; Goering, A W; Ju, K S; McClure, R A; Kemball, J D; Zhukovsky, S; Labeda, D P; Thomson, R J; Kelleher, N L; Metcalf, W W Discovery of the Tyrobetaine Natural Products and Their Biosynthetic Gene Cluster via Metabologenomics. Journal Article ACS Chem Biol., 13 (4), pp. 1029-1037, 2018. Abstract | Links | BibTeX | Tags: @article{Parkinson2018, title = {Discovery of the Tyrobetaine Natural Products and Their Biosynthetic Gene Cluster via Metabologenomics.}, author = {Parkinson, E.I. and Tryon, J.H. and Goering, A.W. and Ju, K.S. and McClure, R.A. and Kemball, J.D. and Zhukovsky, S and Labeda, D.P. and Thomson, R.J. and Kelleher, N.L. and Metcalf, W.W.}, url = {https://pubs.acs.org/doi/10.1021/acschembio.7b01089}, doi = {10.1021/acschembio.7b01089}, year = {2018}, date = {2018-04-20}, journal = {ACS Chem Biol.}, volume = {13}, number = {4}, pages = {1029-1037}, abstract = {Natural products (NPs) are a rich source of medicines, but traditional discovery methods are often unsuccessful due to high rates of rediscovery. Genetic approaches for NP discovery are promising, but progress has been slow due to the difficulty of identifying unique biosynthetic gene clusters (BGCs) and poor gene expression. We previously developed the metabologenomics method, which combines genomic and metabolomic data to discover new NPs and their BGCs. Here, we utilize metabologenomics in combination with molecular networking to discover a novel class of NPs, the tyrobetaines: nonribosomal peptides with an unusual trimethylammonium tyrosine residue. The BGC for this unusual class of compounds was identified using metabologenomics and computational structure prediction data. Heterologous expression confirmed the BGC and suggests an unusual mechanism for trimethylammonium formation. Overall, the discovery of the tyrobetaines shows the great potential of metabologenomics combined with molecular networking and computational structure prediction for identifying interesting biosynthetic reactions and novel NPs.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Natural products (NPs) are a rich source of medicines, but traditional discovery methods are often unsuccessful due to high rates of rediscovery. Genetic approaches for NP discovery are promising, but progress has been slow due to the difficulty of identifying unique biosynthetic gene clusters (BGCs) and poor gene expression. We previously developed the metabologenomics method, which combines genomic and metabolomic data to discover new NPs and their BGCs. Here, we utilize metabologenomics in combination with molecular networking to discover a novel class of NPs, the tyrobetaines: nonribosomal peptides with an unusual trimethylammonium tyrosine residue. The BGC for this unusual class of compounds was identified using metabologenomics and computational structure prediction data. Heterologous expression confirmed the BGC and suggests an unusual mechanism for trimethylammonium formation. Overall, the discovery of the tyrobetaines shows the great potential of metabologenomics combined with molecular networking and computational structure prediction for identifying interesting biosynthetic reactions and novel NPs. |
Miley, G P; Rote, J C; Silverman, R B; Kelleher, N L; Thomson, R J Total Synthesis of Tambromycin Enabled by Indole C-H Functionalization. Journal Article Org Lett., 20 (8), pp. 2369-2373, 2018. Abstract | Links | BibTeX | Tags: @article{Miley2018, title = {Total Synthesis of Tambromycin Enabled by Indole C-H Functionalization.}, author = {Miley, G.P. and Rote, J.C. and Silverman, R.B. and Kelleher, N.L. and Thomson, R.J.}, url = {https://pubs.acs.org/doi/10.1021/acs.orglett.8b00700}, doi = {10.1021/acs.orglett.8b00700}, year = {2018}, date = {2018-04-20}, journal = {Org Lett.}, volume = {20}, number = {8}, pages = {2369-2373}, abstract = {The total synthesis of tambromycin (1), a recently isolated tetrapeptide, is reported. This unusual natural product possesses a highly modified tryptophan-derived indole fragment fused to an α-methylserine-derived oxazoline ring, and a unique noncanonical amino acid residue named tambroline (11). A convergent synthesis of tambromycin was achieved by a 13-step route that leveraged recent developments in the field of C-H functionalization to prepare the complex indole fragment, as well as an efficient synthesis of tambroline that featured a diastereoselective amination of homoproline.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The total synthesis of tambromycin (1), a recently isolated tetrapeptide, is reported. This unusual natural product possesses a highly modified tryptophan-derived indole fragment fused to an α-methylserine-derived oxazoline ring, and a unique noncanonical amino acid residue named tambroline (11). A convergent synthesis of tambromycin was achieved by a 13-step route that leveraged recent developments in the field of C-H functionalization to prepare the complex indole fragment, as well as an efficient synthesis of tambroline that featured a diastereoselective amination of homoproline. |
Ntai, I; Fornelli, L; DeHart, C J; Hutton, J E; Doubleday, P F; LeDuc, R D; van Nispen, A J; Fellers, R T; Whiteley, G; Boja, E S; Rodriguez, H; Kelleher, N L Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk. Journal Article Proc Natl Acad Sci USA, 115 (16), pp. 4140-4145, 2018. Abstract | Links | BibTeX | Tags: @article{Ntai2018, title = {Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk.}, author = {Ntai, I and Fornelli, L and DeHart, C.J. and Hutton, J.E. and Doubleday, P.F. and LeDuc, R.D. and van Nispen, A.J. and Fellers, R.T. and Whiteley, G and Boja, E.S. and Rodriguez, H and Kelleher, N.L.}, url = {https://www.pnas.org/content/115/16/4140.long}, doi = {10.1073/pnas.1716122115}, year = {2018}, date = {2018-04-17}, journal = {Proc Natl Acad Sci USA}, volume = {115}, number = {16}, pages = {4140-4145}, abstract = {Mutations of the KRAS gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic KRAS colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Mutations of the KRAS gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic KRAS colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms. |
Kenney, G E; Dassama, L M K; Pandelia, M E; Gizzi, A S; Martinie, R J; Gao, P; DeHart, C J; Schachner, L F; Skinner, O S; Ro, S Y; Zhu, X; Sadek, M; Thomas, P M; Almo, S C; Bollinger, J M; Jr Krebs, C; Kelleher, N L; Rosenzweig, AC The biosynthesis of methanobactin. Journal Article Science, 359 (6382), pp. 1411-1416, 2018. Abstract | Links | BibTeX | Tags: @article{Kenney2018, title = {The biosynthesis of methanobactin.}, author = {Kenney, G.E. and Dassama, L.M.K. and Pandelia, M.E. and Gizzi, A.S. and Martinie, R.J. and Gao, P and DeHart, C.J. and Schachner, L.F. and Skinner, O.S. and Ro, S.Y. and Zhu, X and Sadek, M and Thomas, P.M. and Almo, S.C. and Bollinger, J.M. and Jr, Krebs, C and Kelleher, N.L. and Rosenzweig, AC}, url = {https://science.sciencemag.org/content/359/6382/1411.long}, doi = {10.1126/science.aap9437}, year = {2018}, date = {2018-03-23}, journal = {Science}, volume = {359}, number = {6382}, pages = {1411-1416}, abstract = {Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis. |
Martin, R W; Des Soye, B J; Kwon, Y C; Kay, J; Davis, R G; Thomas, P M; Majewska, N I; Chen, C X; Marcum, R D; Weiss, M G; Stoddart, A E; Amiram, M; Ranji Charna, A K; Patel, J R; Isaacs, F J; Kelleher, N L; Hong, S H; Jewett, MC Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids. Journal Article Nat. Commun., 9 (1), pp. 1203, 2018. Abstract | Links | BibTeX | Tags: @article{Martin2018, title = {Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids.}, author = {Martin, R.W. and Des Soye, B.J. and Kwon, Y.C. and Kay, J and Davis, R.G. and Thomas, P.M. and Majewska, N.I. and Chen, C.X. and Marcum, R.D. and Weiss, M.G. and Stoddart, A.E. and Amiram, M and Ranji Charna, A.K. and Patel, J.R. and Isaacs, F.J. and Kelleher, N.L. and Hong, S.H. and Jewett, MC}, url = {https://www.nature.com/articles/s41467-018-03469-5}, doi = {10.1038/s41467-018-03469-5}, year = {2018}, date = {2018-03-23}, journal = {Nat. Commun.}, volume = {9}, number = {1}, pages = {1203}, abstract = {Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries. |
Davis, R G; Park, H M; Kim, K; Greer, J B; Fellers, R T; LeDuc, R D; Romanova, E V; Rubakhin, S S; Zombeck, J A; Wu, C; Yau, P M; Gao, P; van Nispen, A J; Patrie, S M; Thomas, P M; Sweedler, J V; Rhodes, J S; Kelleher, N L Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains. Journal Article Anal Chem, 90 (6), pp. 3802-3810, 2018. Abstract | Links | BibTeX | Tags: @article{Davis2018, title = {Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains.}, author = {Davis, R.G. and Park, H.M. and Kim, K and Greer, J.B. and Fellers, R.T. and LeDuc, R.D. and Romanova, E.V. and Rubakhin, S.S. and Zombeck, J.A. and Wu, C and Yau, P.M. and Gao, P and van Nispen, A.J. and Patrie, S.M. and Thomas, P.M. and Sweedler, J.V. and Rhodes, J.S. and Kelleher, N.L.}, url = {https://pubs.acs.org/doi/10.1021/acs.analchem.7b04108}, doi = {10.1021/acs.analchem.7b04108}, year = {2018}, date = {2018-03-20}, journal = {Anal Chem}, volume = {90}, number = {6}, pages = {3802-3810}, abstract = {Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains. |
Smith, L M; Kelleher, N L Proteoforms as the next proteomics currency. Journal Article Science, 359 (6380), pp. 1106-1107, 2018. Abstract | Links | BibTeX | Tags: @article{Smith2018, title = {Proteoforms as the next proteomics currency.}, author = {Smith, L.M. and Kelleher, N.L.}, url = {https://science.sciencemag.org/content/359/6380/1106.long}, doi = {10.1126/science.aat1884}, year = {2018}, date = {2018-03-09}, journal = {Science}, volume = {359}, number = {6380}, pages = {1106-1107}, abstract = {Proteoforms—the different forms of proteins produced from the genome with a variety of sequence variations, splice isoforms, and myriad posttranslational modifications (1)—are critical elements in all biological systems (see the figure, left). Yang et al. (2) recently showed that the functions of proteins produced from splice variants from a given gene—different proteoforms—can be as different as those for proteins encoded by entirely different genes. Li et al. (3) showed that splice variants play a central role in modulating complex traits. However, the standard paradigm of proteomic analysis, the “bottom-up” strategy pioneered by Eng and Yates some 20 years ago (4), does not directly identify proteoforms. We argue that proteomic analysis needs to provide the identities and abundances of the proteoforms themselves, rather than just their peptide surrogates. Developing new proteome-wide strategies to accomplish this goal presents a formidable but not insurmountable technological challenge that will benefit the biomedical community.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Proteoforms—the different forms of proteins produced from the genome with a variety of sequence variations, splice isoforms, and myriad posttranslational modifications (1)—are critical elements in all biological systems (see the figure, left). Yang et al. (2) recently showed that the functions of proteins produced from splice variants from a given gene—different proteoforms—can be as different as those for proteins encoded by entirely different genes. Li et al. (3) showed that splice variants play a central role in modulating complex traits. However, the standard paradigm of proteomic analysis, the “bottom-up” strategy pioneered by Eng and Yates some 20 years ago (4), does not directly identify proteoforms. We argue that proteomic analysis needs to provide the identities and abundances of the proteoforms themselves, rather than just their peptide surrogates. Developing new proteome-wide strategies to accomplish this goal presents a formidable but not insurmountable technological challenge that will benefit the biomedical community. |
LeDuc, R; Schwammle, V; Shortreed, MR ; Cesnick, AJ ; Solntsev, SK ; Shaw, JB ; Martin, MJ ; Vizcaino, JA ; Alpi, E; Danis, P; Kelleher, NL ; Smith, LM ; Ge, Y; Agar, JN ; Chamot-Rooke, J; Loo, JA ; Pasa-Tolic, L; Tsybin, YO ProForma: A Standard Proteoform Notation. Journal Article J Proteome Res., 17 (3), pp. 1321-1325, 2018. Abstract | Links | BibTeX | Tags: @article{LeDuc2018, title = {ProForma: A Standard Proteoform Notation.}, author = {LeDuc, R and Schwammle, V and Shortreed, MR and Cesnick, AJ and Solntsev, SK and Shaw, JB and Martin, MJ and Vizcaino, JA and Alpi,E and Danis, P and Kelleher, NL and Smith, LM and Ge, Y and Agar, JN and Chamot-Rooke, J and Loo, JA and Pasa-Tolic, L and Tsybin, YO}, url = {https://pubs.acs.org/doi/10.1021/acs.jproteome.7b00851}, doi = {10.1021/acs.jproteome.7b00851}, year = {2018}, date = {2018-03-02}, journal = {J Proteome Res.}, volume = {17}, number = {3}, pages = {1321-1325}, abstract = {The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human-readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human-readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard. |
Aebersold, R; Agar, JN ; Amster, IJ ; Baker, MS ; Bertozzi, CR ; Boja, ES ; Costello, CE ; Cravatt, BF ; Fenselau, C; Garcia, BA ; Ge, Y; Gunawardena, J; Hendrickson, RC ; Hergenrother, PJ ; Huber, CG ; Ivanov, AR ; Jensen, ON ; Jewett, MC ; Kelleher, NL ; Kiessling, LL ; Krogan, NJ ; Larsen, MR ; Loo, JA ; Ogorzalek, Loo RR ; Lundberg, E; MacCoss, MJ ; Mallick, P; Mootha, VK ; Mrksich, M; Muir, TW ; Patrie, SM ; Pesavento, JJ ; Pitteri, SJ ; Rodriguez, H; Saghatelian, A; Sandoval, W; Schlüter, H; Sechi, S; Slavoff, SA ; Smith, LM ; Snyder, MP ; Thomas, PM ; Uhlén, M; Van Eyk, JE ; Vidal, M; Walt, DR ; White, FM ; Williams, ER ; Wohlschlager, T; Wysocki, VH ; Yates, NA ; Young, NL ; Zhang, B How many human proteoforms are there? Journal Article Nat Chem Biol., 14 (3), pp. 206-214, 2018. Abstract | Links | BibTeX | Tags: @article{Aebersold2018, title = {How many human proteoforms are there?}, author = {Aebersold, R and Agar, JN and Amster, IJ and Baker, MS and Bertozzi, CR and Boja, ES and Costello, CE and Cravatt, BF and Fenselau, C and Garcia, BA and Ge, Y and Gunawardena, J and Hendrickson, RC and Hergenrother, PJ and Huber, CG and Ivanov, AR and Jensen, ON and Jewett, MC and Kelleher, NL and Kiessling, LL and Krogan, NJ and Larsen, MR and Loo, JA and Ogorzalek, Loo RR and Lundberg, E and MacCoss, MJ and Mallick, P and Mootha, VK and Mrksich, M and Muir, TW and Patrie, SM and Pesavento, JJ and Pitteri, SJ and Rodriguez, H and Saghatelian, A and Sandoval, W and Schlüter, H and Sechi, S and Slavoff, SA and Smith, LM and Snyder, MP and Thomas, PM and Uhlén, M and Van Eyk, JE and Vidal, M and Walt, DR and White, FM and Williams, ER and Wohlschlager, T and Wysocki, VH and Yates, NA and Young, NL and Zhang, B}, url = {https://www.nature.com/articles/nchembio.2576}, doi = {10.1038/nchembio.2576}, year = {2018}, date = {2018-02-14}, journal = {Nat Chem Biol.}, volume = {14}, number = {3}, pages = {206-214}, abstract = {Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype. |
