{"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":12007,"journal":{"name":"European Journal of Mass Spectrometry","volume":null,"pages":null},"PeriodicalIF":1.1000,"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\":12007,\"journal\":{\"name\":\"European Journal of Mass Spectrometry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.1000,\"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\":\"European Journal of Mass Spectrometry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1177/14690667231202695\",\"RegionNum\":4,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2023/9/26 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q4\",\"JCRName\":\"PHYSICS, ATOMIC, MOLECULAR & CHEMICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"European Journal of Mass Spectrometry","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1177/14690667231202695","RegionNum":4,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/9/26 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"PHYSICS, ATOMIC, MOLECULAR & CHEMICAL","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:
JMS - European Journal of Mass Spectrometry, is a peer-reviewed journal, devoted to the publication of innovative research in mass spectrometry. Articles in the journal come from proteomics, metabolomics, petroleomics and other areas developing under the umbrella of the “omic revolution”.