Published online 11 January 2007; doi:10.1038/nprot.2006.427
Shao-En Ong1 & Matthias Mann2
1The Broad Institute of MITand Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.
2Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany. Correspondence should be addressed to S.-E.O. (firstname.lastname@example.org)
Stable isotope labeling by amino acids in cell culture (SILAC) is a simple, robust, yet powerful approach in mass spectrometry (MS)-based quantitative proteomics. SILAC labels cellular proteomes through normal metabolic processes, incorporating non-radioactive, stable isotope-containing amino acids in newly synthesized proteins. Growth medium is prepared where natural (‘‘light’’) amino acids are replaced by ‘‘heavy’’ SILAC amino acids. Cells grown in this medium incorporate the heavy amino acids after five cell doublings and SILAC amino acids have no effect on cell morphology or growth rates. When light and heavy cell populations are mixed, they remain distinguishable by MS, and protein abundances are determined from the relative MS signal intensities. SILAC provides accurate relative quantification without any chemical derivatization or manipulation and enables development of elegant functional assays in proteomics. In this protocol, we describe how to apply SILAC and the use of nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry for protein identification and quantification. This procedure can be completed in 8 days.