Lester S. Manly, , , Anne M. Roberts, , , Joseph S. Beckman, , and , Blaine R. Roberts*,
{"title":"电子捕获解离在色谱时间尺度上发现多肽和小蛋白自上而下的蛋白质组学。","authors":"Lester S. Manly, , , Anne M. Roberts, , , Joseph S. Beckman, , and , Blaine R. Roberts*, ","doi":"10.1021/jasms.5c00116","DOIUrl":null,"url":null,"abstract":"<p >Bottom-up proteomics introduces proteoform ambiguity due to the loss of connectivity between peptides and their original proteoforms. Top-down proteomics (TDP) removes the ambiguity through the direct identification and characterization of intact proteoforms and their respective post-translational modifications (PTM). Electron capture dissociation (ECD) is an efficient and gentle peptide and protein fragmentation strategy that can be used for both bottom-up and top-down approaches. Here, we used an Agilent 6550 Q-TOF mass spectrometer retrofitted with an e-MSion ECD cell. Top-down sequencing capabilities of the cell were evaluated by sequencing of intact peptides and proteins on high-performance liquid chromatography (HPLC) time scales. Amyloid beta 1-40 (Aβ40) was first tested due to its pathophysiological role in Alzheimer’s disease and served as our large peptide standard. We sequenced Aβ40 via reverse-phase HPLC-MS and achieved 95% sequence coverage on chromatographic time scales utilizing a data-dependent acquisition (DDA)-based method. Acetone-precipitated protein extracts from human brain were then separated by HPLC and analyzed with a DDA method, which identified 16 proteoforms between 2 and 17 kDa with sequence coverage ranging from 7 to 90% based on proteoform size and composition. In addition to proteoform identification, ECD fragmentation distinguished multiple isoaspartate modifications from aspartate, as well as accurately differentiating leucine from isoleucine residues directly from the human brain sample. Here, we observed isoaspartate within a thymosin beta-4 proteoform. Additionally, we demonstrated the differentiation of leucine and isoleucine within a subunit of ubiquitin. This study advances the application of LC-Q-TOF instrumentation for discovery-based top-down proteomics utilizing ECD as enabled by the e-MSion ECD cell.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2079–2093"},"PeriodicalIF":2.7000,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/jasms.5c00116","citationCount":"0","resultStr":"{\"title\":\"Electron Capture Dissociation for Discovery Top-Down Proteomics of Peptides and Small Proteins on Chromatographic Time Scales\",\"authors\":\"Lester S. Manly, , , Anne M. Roberts, , , Joseph S. Beckman, , and , Blaine R. Roberts*, \",\"doi\":\"10.1021/jasms.5c00116\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >Bottom-up proteomics introduces proteoform ambiguity due to the loss of connectivity between peptides and their original proteoforms. Top-down proteomics (TDP) removes the ambiguity through the direct identification and characterization of intact proteoforms and their respective post-translational modifications (PTM). Electron capture dissociation (ECD) is an efficient and gentle peptide and protein fragmentation strategy that can be used for both bottom-up and top-down approaches. Here, we used an Agilent 6550 Q-TOF mass spectrometer retrofitted with an e-MSion ECD cell. Top-down sequencing capabilities of the cell were evaluated by sequencing of intact peptides and proteins on high-performance liquid chromatography (HPLC) time scales. Amyloid beta 1-40 (Aβ40) was first tested due to its pathophysiological role in Alzheimer’s disease and served as our large peptide standard. We sequenced Aβ40 via reverse-phase HPLC-MS and achieved 95% sequence coverage on chromatographic time scales utilizing a data-dependent acquisition (DDA)-based method. Acetone-precipitated protein extracts from human brain were then separated by HPLC and analyzed with a DDA method, which identified 16 proteoforms between 2 and 17 kDa with sequence coverage ranging from 7 to 90% based on proteoform size and composition. In addition to proteoform identification, ECD fragmentation distinguished multiple isoaspartate modifications from aspartate, as well as accurately differentiating leucine from isoleucine residues directly from the human brain sample. Here, we observed isoaspartate within a thymosin beta-4 proteoform. Additionally, we demonstrated the differentiation of leucine and isoleucine within a subunit of ubiquitin. 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Electron Capture Dissociation for Discovery Top-Down Proteomics of Peptides and Small Proteins on Chromatographic Time Scales
Bottom-up proteomics introduces proteoform ambiguity due to the loss of connectivity between peptides and their original proteoforms. Top-down proteomics (TDP) removes the ambiguity through the direct identification and characterization of intact proteoforms and their respective post-translational modifications (PTM). Electron capture dissociation (ECD) is an efficient and gentle peptide and protein fragmentation strategy that can be used for both bottom-up and top-down approaches. Here, we used an Agilent 6550 Q-TOF mass spectrometer retrofitted with an e-MSion ECD cell. Top-down sequencing capabilities of the cell were evaluated by sequencing of intact peptides and proteins on high-performance liquid chromatography (HPLC) time scales. Amyloid beta 1-40 (Aβ40) was first tested due to its pathophysiological role in Alzheimer’s disease and served as our large peptide standard. We sequenced Aβ40 via reverse-phase HPLC-MS and achieved 95% sequence coverage on chromatographic time scales utilizing a data-dependent acquisition (DDA)-based method. Acetone-precipitated protein extracts from human brain were then separated by HPLC and analyzed with a DDA method, which identified 16 proteoforms between 2 and 17 kDa with sequence coverage ranging from 7 to 90% based on proteoform size and composition. In addition to proteoform identification, ECD fragmentation distinguished multiple isoaspartate modifications from aspartate, as well as accurately differentiating leucine from isoleucine residues directly from the human brain sample. Here, we observed isoaspartate within a thymosin beta-4 proteoform. Additionally, we demonstrated the differentiation of leucine and isoleucine within a subunit of ubiquitin. This study advances the application of LC-Q-TOF instrumentation for discovery-based top-down proteomics utilizing ECD as enabled by the e-MSion ECD cell.
期刊介绍:
The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role.
Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives