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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of SILAC Media

  Materials
  • 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

  Materials
  • 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, http://www.proxeon.com, 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

  Materials
  • 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)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Aebersold, R. and Mann, M. 2003. Mass spectrometry‐based proteomics. Nature 422:198‐207.
   Ambler, R.P. and Rees, M.W. 1959. Epsilon‐N‐Methyl‐lysine in bacterial flagellar protein. Nature 184:56‐57.
   Bannister, A.J. and Kouzarides, T. 2005. Reversing histone methylation. Nature 436:1103‐1106.
   Bedford, M.T. and Richard, S. 2005. Arginine methylation an emerging regulator of protein function. Mol. Cell 18:263‐272.
   Brame, C.J., Moran, M.F., and McBroom‐Cerajewski, L.D. 2004. A mass spectrometry based method for distinguishing between symmetrically and asymmetrically dimethylated arginine residues. Rapid Commun. Mass Spectrom. 18:877‐881.
   Cuthbert, G.L., Daujat, S., Snowden, A.W., Erdjument‐Bromage, H., Hagiwara, T., Yamada, M., Schneider, R., Gregory, P.D., Tempst, P., Bannister, A.J., and Kouzarides, T. 2004. Histone deimination antagonizes arginine methylation. Cell 118:545‐553.
   Fackelmayer, F.O. 2005. Protein arginine methyltransferases: Guardians of the Arg? Trends Biochem. Sci. 30:666‐671.
   Gehrmann, M.L., Hathout, Y., and Fenselau, C. 2004. Evaluation of metabolic labeling for comparative proteomics in breast cancer cells. J. Proteome Res. 3:1063‐1068.
   Gruhler, A., Olsen, J.V., Mohammed, S., Mortensen, P., Faergeman, N.J., Mann, M., and Jensen, O.N. 2005a. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. Mol. Cell. Proteomics 4:310‐327.
   Gruhler, A., Schulze, W.X., Matthiesen, R., Mann, M., and Jensen, O.N. 2005b. Stable isotope labeling of Arabidopsis thaliana cells and quantitative proteomics by mass spectrometry. Mol. Cell. Proteomics 4:1697‐1709.
   Ibarrola, N., Kalume, D.E., Gronborg, M., Iwahori, A., and Pandey, A. 2003. A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture. Anal. Chem. 75:6043‐6049.
   Ishihama, Y. 2005. Proteomic LC‐MS systems using nanoscale liquid chromatography with tandem mass spectrometry. J. Chromatogr. A. 1067:73‐83.
   Mann, M. and Jensen, O.N. 2003. Proteomic analysis of post‐translational modifications. Nat. Biotechnol. 21:255‐261.
   McBride, A.E. and Silver, P.A. 2001. State of the arg: Protein methylation at arginine comes of age. Cell 106:5‐8.
   Nirmalan, N., Sims, P.F., and Hyde, J.E. 2004. Quantitative proteomics of the human malaria parasite Plasmodium falciparum and its application to studies of development and inhibition. Mol. Microbiol. 52:1187‐1199.
   Olsen, J.V., Ong, S.E., and Mann, M. 2004. Trypsin cleaves exclusively C‐terminal to arginine and lysine residues. Mol. Cell. Proteomics 3:608‐614.
   Ong, S.E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A., and Mann, M. 2002. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1:376‐386.
   Ong, S.E., Kratchmarova, I., and Mann, M. 2003. Properties of 13C‐substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC). J. Proteome Res. 2:173‐181.
   Ong, S.E. and Mann, M. 2005. Mass spectrometry‐based proteomics turns quantitative. Nat. Chem. Biol. 1:252‐262.
   Ong, S.E., Mittler, G., and Mann, M. 2004. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC. Nat. Methods 1:119‐126.
   Peng, J. and Gygi, S.P. 2001. Proteomics: The move to mixtures. J. Mass Spectrom. 36:1083‐1091.
   Rappsilber, J., Ishihama, Y., and Mann, M. 2003a. Stop and go extraction tips for matrix‐assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75:663‐670.
   Rappsilber, J., Friesen, W.J., Paushkin, S., Dreyfuss, G., and Mann, M. 2003b. Detection of arginine dimethylated peptides by parallel precursor ion scanning mass spectrometry in positive ion mode. Anal. Chem. 75:3107‐3114.
   Shi, Y., Lan, F., Matson, C., Mulligan, P., Whetstine, J.R., Cole, P.A., Casero, R.A., and Shi, Y. 2004. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119:941‐953.
   Steen, H. and Mann, M. 2004. The ABC's (and XYZ's) of peptide sequencing. Nat. Rev. Mol. Cell. Biol. 5:699‐711.
   Wang, Y., Wysocka, J., Sayegh, J., Lee, Y.H., Perlin, J.R., Leonelli, L., Sonbuchner, L.S., McDonald, C.H., Cook, R.G., Dou, Y., Roeder, R.G., Clarke, S., Stallcup, M.R., Allis, C.D., and Coonrod, S.A. 2004. Human PAD4 regulates histone arginine methylation levels via demethylimination. Science 306:279‐283.
   Zhang, K., Yau, P.M., Chandrasekhar, B., New, R., Kondrat, R., Imai, B.S., and Bradbury, M.E. 2004. Differentiation between peptides containing acetylated or tri‐methylated lysines by mass spectrometry: an application for determining lysine 9 acetylation and methylation of histone H3. Proteomics 4:1‐10.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library