Identifying and Quantifying Sites of Protein Methylation by Heavy Methyl SILAC

Shao‐En Ong1, Matthias Mann2

1 The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, 2 Max Planck Institute for Biochemistry, Martinsried
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.9
DOI:  10.1002/0471140864.ps1409s46
Online Posting Date:  December, 2006
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A new appreciation of protein methylation comes with the recent discovery of demethylases, now placing methylation in the realm of a transient, reversible modification. Classical approaches to study methylation are laborious and involve radioactive, in vitro, enzyme‐substrate labeling experiments with purified proteins. Mass spectrometry–based proteomics allows the unbiased analysis of complex protein mixtures and is increasingly applied to the study of post‐translational modifications. However, it is particularly challenging to study methylation by proteomics because of the number of residues affected and the degree of methylation that can occur. Heavy methyl SILAC is a metabolic labeling strategy that harnesses the cell's machinery to convert a nonradioactive, stable isotope labeled version of methionine into the ‘heavy’ biological methyl donor S‐adenosylmethionine. Cells incorporate this ‘heavy’ methyl group throughout their methylated substrates. This technique increases confidence in identifying and quantifying of sites of protein methylation.

Keywords: SILAC; mass spectrometry; proteomics; metabolic labeling; methylation; quantification; post‐translational modification

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Table of Contents

  • Basic Protocol 1: Preparation of SILAC Media
  • Basic Protocol 2: Testing for full Incorporation of SILAC Amino Acid
  • Basic Protocol 3: Studying Protein Methylation with Heavy Methyl SILAC with Antibodies Against Methylated Proteins
  • Support Protocol 1: Data Analysis to Identify and Quantify Methylated Peptides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of SILAC Media

  • SILAC amino acid stock solutions (see reciperecipes)
  • Tissue culture medium appropriate for cells (e.g., Dulbecco's Modified Eagle's Medium, DMEM, appendix 3C; Life Technologies Inc.) deficient in SILAC amino acid of choice (methionine, in this example; custom‐synthesized “drop‐out” amino acid medium is available as a special order product from most medium distributors)
  • L‐methionine, natural abundance form, tissue‐culture‐grade
  • L‐methionine‐13C,2H 3 (methyl‐13C, 2H 3), stable isotope enriched form, where stable isotopes are located in the methyl group on the side chain of methionine (Sigma‐Isotec)
  • Antibiotics for cell culture, e.g., penicillin and streptomycin
  • Glutamine
  • Dialyzed serum (Invitrogen)
  • 0.2‐µm pore size filter
  • Tissue‐culture‐grade filter bottles, sterile
NOTE: All reagents and equipment coming in contact with live cells must be sterile and standard sterile tissue culture technique should be used.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: Medium that is deficient in multiple amino acids should always be reconstituted to its original basal formulation as prescribed by manufacturer's instructions.

Basic Protocol 2: Testing for full Incorporation of SILAC Amino Acid

  • Cells
  • SILAC media from protocol 1
  • Tissue‐culture‐grade trypsin for lifting adherent cells
  • Urea lysis buffer or a mass spectrometry‐compatible lysis buffer for in‐solution proteolytic digest of proteins.
  • 1 M dithiothreitol
  • 600 mM iodoacetamide
  • Endoproteinase Lys‐C, proteomics grade (Roche Diagnostics)
  • 50 mM ammonium bicarbonate
  • Trypsin, proteomics grade
  • Formic acid
  • C18 reversed phase micro‐columns (StageTips; Proxeon Biosystems,, Rappsilber et al., )
  • Additional reagents and equipment for standard tissue cell culture and cell harvesting ( appendix 3C) and mass spectrometry (Chapter 16)

Basic Protocol 3: Studying Protein Methylation with Heavy Methyl SILAC with Antibodies Against Methylated Proteins

  • Cells fully adapted to ‘light’ and ‘heavy’ SILAC medium as described in protocol 2
  • Lysis buffer (see recipe) compatible with co‐immunoprecipitation experiments
  • Trypsin, tissue‐culture‐grade
  • Protein A or G agarose beads (Pierce or Sigma)
  • Antibodies directed against a specific protein or a class of methylated residues, such as anti‐methylarginine or anti‐methyl‐lysine antibodies
  • 2× or 6× SDS‐PAGE loading buffer (see recipe)
  • 50 mM glycine, pH 2.4
  • Cell scraper
  • Econo‐Pac chromatography column (Bio‐Rad)
  • Additional reagents and equipment for adapting cells to SILAC media ( protocol 2), Bradford protein assay (unit 3.4), 1‐D SDS‐PAGE (unit 10.1), staining of gels (unit 10.5), and mass spectrometry (Chapter 16)
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Literature Cited

Literature Cited
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