{"title":"用于蛋白质足迹自上而下分析的MALDI源内衰变(ISD)方法。","authors":"Ruidong Jiang, Don L Rempel, Michael L Gross","doi":"10.1177/14690667231202695","DOIUrl":null,"url":null,"abstract":"<p><p>Irreversible protein footprinting is a mass spectrometry-based approach in which solvent-accessible sites of a protein are modified to assess high-order protein structure. Structural insights can be gained by determining the position and extents of modification. The usual approach to obtain the \"footprint\" is to analyze the protein through bottom-up LC-MS/MS. In this approach, the proteins are digested to yield a mixture of peptides that are then separated by LC before locating the modification sites by MS/MS. This process consumes substantial amounts of time and is difficult to accelerate for applications that require quick and high-throughput analysis. Here, we describe employing matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) to analyze a footprinted small test protein (ubiquitin) via a top-down approach. Matrix-assisted laser desorption/ionization is easily adapted for high-throughput analysis, and top-down strategies can avoid lengthy proteolysis and LC separation. We optimized the method with model peptides and then demonstrated its feasibility on ubiquitin submitted to two types of footprinting. We found that MALDI ISD can produce a comprehensive set of fragment ions for small proteins, affording footprinting information in a fast manner and giving results that agree with the established methods, and serve as a rough measure of protein solvent accessibility. To assist in the implementation of the MALDI approach, we developed a method of processing top-down ISD data.</p>","PeriodicalId":1,"journal":{"name":"Accounts of Chemical Research","volume":null,"pages":null},"PeriodicalIF":16.4000,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11092977/pdf/","citationCount":"0","resultStr":"{\"title\":\"Toward a MALDI in-source decay (ISD) method for top-down analysis of protein footprinting.\",\"authors\":\"Ruidong Jiang, Don L Rempel, Michael L Gross\",\"doi\":\"10.1177/14690667231202695\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Irreversible protein footprinting is a mass spectrometry-based approach in which solvent-accessible sites of a protein are modified to assess high-order protein structure. Structural insights can be gained by determining the position and extents of modification. The usual approach to obtain the \\\"footprint\\\" is to analyze the protein through bottom-up LC-MS/MS. In this approach, the proteins are digested to yield a mixture of peptides that are then separated by LC before locating the modification sites by MS/MS. This process consumes substantial amounts of time and is difficult to accelerate for applications that require quick and high-throughput analysis. Here, we describe employing matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) to analyze a footprinted small test protein (ubiquitin) via a top-down approach. Matrix-assisted laser desorption/ionization is easily adapted for high-throughput analysis, and top-down strategies can avoid lengthy proteolysis and LC separation. We optimized the method with model peptides and then demonstrated its feasibility on ubiquitin submitted to two types of footprinting. We found that MALDI ISD can produce a comprehensive set of fragment ions for small proteins, affording footprinting information in a fast manner and giving results that agree with the established methods, and serve as a rough measure of protein solvent accessibility. To assist in the implementation of the MALDI approach, we developed a method of processing top-down ISD data.</p>\",\"PeriodicalId\":1,\"journal\":{\"name\":\"Accounts of Chemical Research\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":16.4000,\"publicationDate\":\"2023-10-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11092977/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Accounts of Chemical Research\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1177/14690667231202695\",\"RegionNum\":1,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2023/9/26 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q1\",\"JCRName\":\"CHEMISTRY, MULTIDISCIPLINARY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Accounts of Chemical Research","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1177/14690667231202695","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/9/26 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
Toward a MALDI in-source decay (ISD) method for top-down analysis of protein footprinting.
Irreversible protein footprinting is a mass spectrometry-based approach in which solvent-accessible sites of a protein are modified to assess high-order protein structure. Structural insights can be gained by determining the position and extents of modification. The usual approach to obtain the "footprint" is to analyze the protein through bottom-up LC-MS/MS. In this approach, the proteins are digested to yield a mixture of peptides that are then separated by LC before locating the modification sites by MS/MS. This process consumes substantial amounts of time and is difficult to accelerate for applications that require quick and high-throughput analysis. Here, we describe employing matrix-assisted laser desorption/ionization (MALDI) in-source decay (ISD) to analyze a footprinted small test protein (ubiquitin) via a top-down approach. Matrix-assisted laser desorption/ionization is easily adapted for high-throughput analysis, and top-down strategies can avoid lengthy proteolysis and LC separation. We optimized the method with model peptides and then demonstrated its feasibility on ubiquitin submitted to two types of footprinting. We found that MALDI ISD can produce a comprehensive set of fragment ions for small proteins, affording footprinting information in a fast manner and giving results that agree with the established methods, and serve as a rough measure of protein solvent accessibility. To assist in the implementation of the MALDI approach, we developed a method of processing top-down ISD data.
期刊介绍:
Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance.
Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.