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{"title":"选择性富集结合蛋白质组学鉴定拟南芥s -酰化质膜蛋白","authors":"Lijuan Zhou, Mowei Zhou, Marina A. Gritsenko, Gary Stacey","doi":"10.1002/cppb.20119","DOIUrl":null,"url":null,"abstract":"<p>Protein S-acylation, predominately in the form of palmitoylation, is a reversible lipid post-translational modification on cysteines that plays important roles in protein localization, trafficking, activity, and complex assembly. The functions and regulatory mechanisms of S-acylation have been extensively studied in mammals owing to remarkable development of high-resolution proteomics and the discovery of the S-acylation-related enzymes. However, the advancement of S-acylation studies in plants lags behind that in mammals, mainly due to the lack of knowledge about proteins responsible for this process, such as protein acyltransferases and their substrates. In this article, a set of systematic protocols to study global S-acylation in <i>Arabidopsis</i> seedlings is described. The procedures are presented in detail, including preparation of <i>Arabidopsis</i> seedlings, enrichment of plasma membrane (PM) proteins, ensuing enrichment of S-acylated proteins/peptides based on the acyl-biotin exchange method, and large-scale identification of S-acylated proteins/peptides via mass spectrometry. This approach enables researchers to study S-acylation of PM proteins in plants in a systematic and straightforward way. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preparation of <i>Arabidopsis</i> seedling materials</p><p><b>Basic Protocol 2</b>: Isolation and enrichment of plasma membrane proteins</p><p><b>Support Protocol 1</b>: Determination of protein concentration using BCA assay</p><p><b>Basic Protocol 3</b>: Enrichment of S-acylated proteins by acyl-biotin exchange method</p><p><b>Support Protocol 2</b>: Protein precipitation by methanol/chloroform method</p><p><b>Basic Protocol 4</b>: Trypsin digestion and proteomic analysis</p><p><b>Alternate Protocol</b>: Pre-resin digestion and peptide-level enrichment</p>","PeriodicalId":10932,"journal":{"name":"Current protocols in plant biology","volume":"5 4","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2020-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cppb.20119","citationCount":"2","resultStr":"{\"title\":\"Selective Enrichment Coupled with Proteomics to Identify S-Acylated Plasma Membrane Proteins in Arabidopsis\",\"authors\":\"Lijuan Zhou, Mowei Zhou, Marina A. 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The procedures are presented in detail, including preparation of <i>Arabidopsis</i> seedlings, enrichment of plasma membrane (PM) proteins, ensuing enrichment of S-acylated proteins/peptides based on the acyl-biotin exchange method, and large-scale identification of S-acylated proteins/peptides via mass spectrometry. 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Selective Enrichment Coupled with Proteomics to Identify S-Acylated Plasma Membrane Proteins in Arabidopsis
Protein S-acylation, predominately in the form of palmitoylation, is a reversible lipid post-translational modification on cysteines that plays important roles in protein localization, trafficking, activity, and complex assembly. The functions and regulatory mechanisms of S-acylation have been extensively studied in mammals owing to remarkable development of high-resolution proteomics and the discovery of the S-acylation-related enzymes. However, the advancement of S-acylation studies in plants lags behind that in mammals, mainly due to the lack of knowledge about proteins responsible for this process, such as protein acyltransferases and their substrates. In this article, a set of systematic protocols to study global S-acylation in Arabidopsis seedlings is described. The procedures are presented in detail, including preparation of Arabidopsis seedlings, enrichment of plasma membrane (PM) proteins, ensuing enrichment of S-acylated proteins/peptides based on the acyl-biotin exchange method, and large-scale identification of S-acylated proteins/peptides via mass spectrometry. This approach enables researchers to study S-acylation of PM proteins in plants in a systematic and straightforward way. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : Preparation of Arabidopsis seedling materials
Basic Protocol 2 : Isolation and enrichment of plasma membrane proteins
Support Protocol 1 : Determination of protein concentration using BCA assay
Basic Protocol 3 : Enrichment of S-acylated proteins by acyl-biotin exchange method
Support Protocol 2 : Protein precipitation by methanol/chloroform method
Basic Protocol 4 : Trypsin digestion and proteomic analysis
Alternate Protocol : Pre-resin digestion and peptide-level enrichment
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