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Title: 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 inArabidopsisseedlings is described. The procedures are presented in detail, including preparation ofArabidopsisseedlings, 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 ofArabidopsisseedling 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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Author(s) / Creator(s):
 ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Current Protocols in Plant Biology
Medium: X
Sponsoring Org:
National Science Foundation
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    Basic Protocol 1: Isolation of EVs from the apoplastic fluid ofArabidopsis thaliana

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    Basic Protocol 1: Long‐term auxin‐mediated depletion on plates

    Support Protocol: Preparation of NGM and NGM‐auxin plates

    Basic Protocol 2: Rapid auxin‐mediated depletion via soaking

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    Basic Protocol 1: Lentivirus production and expression line creation

    Support Protocol 1: Six‐well assay for estimation of production cell line yield

    Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His‐tags

    Basic Protocol 2: Cultures for production of Class II MHC proteins

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    Alternate Protocol 1: IMAC purification of His‐tagged Class II MHC

    Support Protocol 3: Protein concentration measurements and adjustments

    Support Protocol 4: Polishing purification by anion‐exchange chromatography

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