Linda B Lieu, Joshua D Hinkle, John E P Syka, Luca Fornelli
{"title":"利用离子-离子活化和离子-光子活化改进带有多个二硫键的蛋白质测序:人类血清白蛋白案例研究。","authors":"Linda B Lieu, Joshua D Hinkle, John E P Syka, Luca Fornelli","doi":"10.1021/jasms.4c00391","DOIUrl":null,"url":null,"abstract":"<p><p>Gas-phase sequencing of large intact proteins (>30 kDa) via tandem mass spectrometry is an inherently challenging process that is further complicated by the extensive overlap of multiply charged product ion peaks, often characterized by a low signal-to-noise ratio. Disulfide bonds exacerbate this issue because of the need to cleave both the S-S and backbone bonds to liberate sequence informative fragments. Although electron-based ion activation techniques such as electron transfer dissociation (ETD) have been proven to rupture disulfide bonds in whole protein ions, they still struggle to produce extensive sequencing when multiple, concatenated S-S bonds are present on the same large polypeptide chain. Here, we evaluate the increase in sequence coverage obtained by combining activated-ion ETD (AI-ETD) and proton transfer charge reduction (PTCR) in the analysis of 66 kDa human serum albumin, which holds 17 disulfide bridges. We also describe the combination of AI-ETD with supplemental postactivation of the ETD reaction products via higher-energy collisional dissociation─a hybrid fragmentation method termed AI-EThcD. AI-EThcD leads to a further improvement compared to AI-ETD in both the global number of cleaved backbone bonds and the number of ruptured backbone bonds from disulfide-protected regions. Our results also demonstrate that the full potential of AI-ETD and AI-EThcD is unveiled only when combined with PTCR: reduction in overlap of ion signals leads to a sequence coverage as high as 39% in a single experiment, highlighting the relevance of spectral simplification in top-down mass spectrometry of large proteins.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":"3265-3273"},"PeriodicalIF":3.1000,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Leveraging Ion-Ion and Ion-Photon Activation to Improve the Sequencing of Proteins Carrying Multiple Disulfide Bonds: The Human Serum Albumin Case Study.\",\"authors\":\"Linda B Lieu, Joshua D Hinkle, John E P Syka, Luca Fornelli\",\"doi\":\"10.1021/jasms.4c00391\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Gas-phase sequencing of large intact proteins (>30 kDa) via tandem mass spectrometry is an inherently challenging process that is further complicated by the extensive overlap of multiply charged product ion peaks, often characterized by a low signal-to-noise ratio. Disulfide bonds exacerbate this issue because of the need to cleave both the S-S and backbone bonds to liberate sequence informative fragments. Although electron-based ion activation techniques such as electron transfer dissociation (ETD) have been proven to rupture disulfide bonds in whole protein ions, they still struggle to produce extensive sequencing when multiple, concatenated S-S bonds are present on the same large polypeptide chain. Here, we evaluate the increase in sequence coverage obtained by combining activated-ion ETD (AI-ETD) and proton transfer charge reduction (PTCR) in the analysis of 66 kDa human serum albumin, which holds 17 disulfide bridges. We also describe the combination of AI-ETD with supplemental postactivation of the ETD reaction products via higher-energy collisional dissociation─a hybrid fragmentation method termed AI-EThcD. AI-EThcD leads to a further improvement compared to AI-ETD in both the global number of cleaved backbone bonds and the number of ruptured backbone bonds from disulfide-protected regions. Our results also demonstrate that the full potential of AI-ETD and AI-EThcD is unveiled only when combined with PTCR: reduction in overlap of ion signals leads to a sequence coverage as high as 39% in a single experiment, highlighting the relevance of spectral simplification in top-down mass spectrometry of large proteins.</p>\",\"PeriodicalId\":672,\"journal\":{\"name\":\"Journal of the American Society for Mass Spectrometry\",\"volume\":\" \",\"pages\":\"3265-3273\"},\"PeriodicalIF\":3.1000,\"publicationDate\":\"2024-12-04\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of the American Society for Mass Spectrometry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1021/jasms.4c00391\",\"RegionNum\":2,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/11/7 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of the American Society for Mass Spectrometry","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1021/jasms.4c00391","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/11/7 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Leveraging Ion-Ion and Ion-Photon Activation to Improve the Sequencing of Proteins Carrying Multiple Disulfide Bonds: The Human Serum Albumin Case Study.
Gas-phase sequencing of large intact proteins (>30 kDa) via tandem mass spectrometry is an inherently challenging process that is further complicated by the extensive overlap of multiply charged product ion peaks, often characterized by a low signal-to-noise ratio. Disulfide bonds exacerbate this issue because of the need to cleave both the S-S and backbone bonds to liberate sequence informative fragments. Although electron-based ion activation techniques such as electron transfer dissociation (ETD) have been proven to rupture disulfide bonds in whole protein ions, they still struggle to produce extensive sequencing when multiple, concatenated S-S bonds are present on the same large polypeptide chain. Here, we evaluate the increase in sequence coverage obtained by combining activated-ion ETD (AI-ETD) and proton transfer charge reduction (PTCR) in the analysis of 66 kDa human serum albumin, which holds 17 disulfide bridges. We also describe the combination of AI-ETD with supplemental postactivation of the ETD reaction products via higher-energy collisional dissociation─a hybrid fragmentation method termed AI-EThcD. AI-EThcD leads to a further improvement compared to AI-ETD in both the global number of cleaved backbone bonds and the number of ruptured backbone bonds from disulfide-protected regions. Our results also demonstrate that the full potential of AI-ETD and AI-EThcD is unveiled only when combined with PTCR: reduction in overlap of ion signals leads to a sequence coverage as high as 39% in a single experiment, highlighting the relevance of spectral simplification in top-down mass spectrometry of large proteins.
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