{"title":"利用目标片段匹配提高自上而下的序列覆盖率","authors":"Matthew T Robey, Kenneth R Durbin","doi":"10.1021/jasms.4c00161","DOIUrl":null,"url":null,"abstract":"<p><p>Top-down mass spectrometry (TDMS) of intact proteins and antibodies enables direct determination of truncations, sequence variants, post-translational modifications, and disulfides without the need for any proteolytic cleavage. While mass deconvolution of top-down tandem mass spectra is typically used to identify fragment masses for matching to candidate proteoforms, larger molecules such as monoclonal antibodies can produce many fragment ions, making spectral interpretation challenging. Here, we explore an alternative approach for proteoform spectral matching that is better suited for larger protein analysis. This workflow uses direct matching of theoretical proteoform isotopic distributions to TDMS spectra, avoiding drawbacks of mass deconvolution such as poor sensitivity and problems differentiating overlapping distributions. Using a data set that analyzed an intact NIST monoclonal antibody across different fragmentation modes, we show that this isotope fitting strategy increased the sequence coverage of both light and heavy chain sequences >3-fold. We further found that isotope fitting is particularly amenable to identifying large fragments, including those near the hinge region that have been traditionally difficult to analyze by top-down methods. These advances in proteoform spectral matching can greatly increase the power of top-down analyses for intact biotherapeutics and other large molecules.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":"3296-3300"},"PeriodicalIF":3.1000,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11623164/pdf/","citationCount":"0","resultStr":"{\"title\":\"Improving Top-Down Sequence Coverage with Targeted Fragment Matching.\",\"authors\":\"Matthew T Robey, Kenneth R Durbin\",\"doi\":\"10.1021/jasms.4c00161\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Top-down mass spectrometry (TDMS) of intact proteins and antibodies enables direct determination of truncations, sequence variants, post-translational modifications, and disulfides without the need for any proteolytic cleavage. While mass deconvolution of top-down tandem mass spectra is typically used to identify fragment masses for matching to candidate proteoforms, larger molecules such as monoclonal antibodies can produce many fragment ions, making spectral interpretation challenging. Here, we explore an alternative approach for proteoform spectral matching that is better suited for larger protein analysis. This workflow uses direct matching of theoretical proteoform isotopic distributions to TDMS spectra, avoiding drawbacks of mass deconvolution such as poor sensitivity and problems differentiating overlapping distributions. Using a data set that analyzed an intact NIST monoclonal antibody across different fragmentation modes, we show that this isotope fitting strategy increased the sequence coverage of both light and heavy chain sequences >3-fold. We further found that isotope fitting is particularly amenable to identifying large fragments, including those near the hinge region that have been traditionally difficult to analyze by top-down methods. These advances in proteoform spectral matching can greatly increase the power of top-down analyses for intact biotherapeutics and other large molecules.</p>\",\"PeriodicalId\":672,\"journal\":{\"name\":\"Journal of the American Society for Mass Spectrometry\",\"volume\":\" \",\"pages\":\"3296-3300\"},\"PeriodicalIF\":3.1000,\"publicationDate\":\"2024-12-04\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11623164/pdf/\",\"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.4c00161\",\"RegionNum\":2,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/10/22 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.4c00161","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/10/22 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Improving Top-Down Sequence Coverage with Targeted Fragment Matching.
Top-down mass spectrometry (TDMS) of intact proteins and antibodies enables direct determination of truncations, sequence variants, post-translational modifications, and disulfides without the need for any proteolytic cleavage. While mass deconvolution of top-down tandem mass spectra is typically used to identify fragment masses for matching to candidate proteoforms, larger molecules such as monoclonal antibodies can produce many fragment ions, making spectral interpretation challenging. Here, we explore an alternative approach for proteoform spectral matching that is better suited for larger protein analysis. This workflow uses direct matching of theoretical proteoform isotopic distributions to TDMS spectra, avoiding drawbacks of mass deconvolution such as poor sensitivity and problems differentiating overlapping distributions. Using a data set that analyzed an intact NIST monoclonal antibody across different fragmentation modes, we show that this isotope fitting strategy increased the sequence coverage of both light and heavy chain sequences >3-fold. We further found that isotope fitting is particularly amenable to identifying large fragments, including those near the hinge region that have been traditionally difficult to analyze by top-down methods. These advances in proteoform spectral matching can greatly increase the power of top-down analyses for intact biotherapeutics and other large molecules.
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