Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics

• DOI: 10.1074/mcp.m200025-mcp200
• Publication Date: 2003-04-02T21:49:56Z
• Authors: Shao-En Ong; Blagoy Blagoev; Irina Kratchmarova; Dan Bach Kristensen; Hanno Steen; Akhilesh Pandey; Matthias Mann
• Keywords: Proteome; Isobaric labeling; Label-free Quantification

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification of complex protein mixtures. Here we describe a method, termed SILAC, labeling amino acids in cell culture, vivo incorporation specific into all mammalian proteins. Mammalian lines are grown media lacking standard essential acid supplemented with non-radioactive, isotopically labeled form that acid, this case deuterated leucine (Leu-d3). We find growth cells maintained these is no different from normal as evidenced morphology, doubling time, ability to differentiate. Complete Leu-d3 occurred after five doublings proteins studied. Protein populations experimental control samples mixed directly harvesting, straightforward every leucine-containing peptide incorporates either or Leu-d3. applied technique relative changes expression during process muscle differentiation. Proteins were found be up-regulated include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, pyruvate kinase M2. SILAC simple, inexpensive, accurate procedure can used quantitative proteomic approach any culture system. Proteomics, large scale study complement tissue, its origins technology (2D) 1The abbreviations are: 2D, two-dimensional; ICAT, isotope-coded affinity tag; MS, spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS/MS, tandem MS; 1D, one-dimensional. electrophoresis invented more than 25 years ago (1.O’Farrell P.H. High resolution proteins.J. Biol. Chem. 1975; 250: 4007-4021Google Scholar, 2.Klose J. Kobalz U. 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