Mass‐Tag Labeling Using Acyl‐PEG Exchange for the Determination of Endogenous Protein S‐Fatty Acylation

Avital Percher1, Emmanuelle Thinon1, Howard Hang1

1 Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.17
DOI:  10.1002/cpps.36
Online Posting Date:  August, 2017
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Abstract

The covalent coupling of fatty acids to proteins provides an important mechanism of regulation in cells. In eukaryotes, cysteine fatty acylation (S‐fatty acylation) has been shown to be critical for protein function in a variety of cellular pathways as well as microbial pathogenesis. While methods developed over the past decade have improved the detection and profiling of S‐fatty acylation, these are hampered in their ability to characterize endogenous protein S‐fatty acylation levels under physiological conditions. Furthermore, understanding the contribution of specific sites and levels of S‐fatty acylation remains a major challenge. To evaluate S‐fatty acylation of endogenous proteins as well as to determine the number of S‐fatty acylation events, we developed the acyl‐PEG exchange (APE) that utilizes cysteine‐specific chemistry to exchange S‐fatty acylation sites with mass‐tags of defined size, which can be readily observed by western blotting. APE provides a readily accessible approach to investigate endogenous S‐fatty acylation from any sample source, with high sensitivity and broad applicability that complements the current toolbox of methods for thioester‐based post‐translational modifications. © 2017 by John Wiley & Sons, Inc.

Keywords: mass‐shift; PEGylation; post‐translational modification quantification; s‐fatty‐acylation

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2E), sterile
  • 1× lysis buffer (see recipe)
  • 0.5 M EDTA, pH 8 (Sigma)
  • Bicinchoninic acid (BCA) assay kit (unit 3.4; Olson and Markwell, )
  • 200 mM neutralized TCEP (see recipe)
  • 1 M N‐ethyl maleimide (NEM; see recipe)
  • Methanol, pre‐chilled on ice
  • Chloroform, pre‐chilled on ice
  • 1× TEA buffer/4% SDS (see recipe)
  • 1× TEA buffer/4% SDS (see recipe) with 4 mM EDTA
  • 1 M neutralized hydroxylamine hydrochloride (NH 2OH) (see recipe)
  • 1× TEA buffer/0.2% Triton X‐100 (see recipe)
  • 5 and 10 kDa methoxypolyethylene glycol maleimide (mPEG‐Mal; see reciperecipes)
  • 1× and 4× Laemmli buffer (see recipe)
  • Anti‐calnexin rabbit polyclonal primary antibody; dilute 1:2,000; Abcam, cat. no. ab22595
  • Goat anti‐rabbit secondary antibody; dilute 1:5000; Calbiochem, cat. no. DC03L
  • 1.5‐ml microcentrifuge tubes (1.5 ml)
  • Refrigerated microcentrifuge
  • Sonicator (e.g., Ultrasonic Cleaner, VWR)
  • Nutating mixer (e.g., Thomas Scientific)
  • 95oC heating block
  • Vacuum evaporator (e.g., Centrivap Concentrator, Labconco)
  • 4–20% Criterion‐TGX Stain Free polyacrylamide gels (Bio‐Rad)
  • Nitrocellulose membrane (0.2 µm)
  • Additional reagents and equipment for trypsinization ( appendix 3C; Phelan, ), BCA protein assay (unit 3.4; Olson and Markwell, ), SDS‐PAGE (unit 10.1; Gallagher, ), and western blotting [unit 10.7 (Goldman, Ursitti, Mozdzanowski, & Speicher, ) and unit 10.8 (Goldman, Harper, & Speicher, )]
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Figures

Videos

Literature Cited

Literature Cited
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