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A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)

Abstract

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.

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Figure 1: Encoding quantitative information into whole proteomes with SILAC.
Figure 2: Experimental workflows in quantitative proteomics.
Figure 3: Overview of SILAC protocol.
Figure 4: Evaluating peptides for quantification.
Figure 5: Quantification of SILAC peptides.

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Acknowledgements

The protocol and suggestions described here represent the experience accumulated from several years of experimentation by a number of researchers in the Mann laboratory. In particular, we gratefully acknowledge Blagoy Blagoev, Jens Andersen, Peter Mortensen, Jesper V. Olsen, Leonard J. Foster and other members of the Center for Experimental BioInformatics (CEBI) at the University of Southern Denmark. We also thank the present members of the Proteomics group at the Broad Institute of MIT and Harvard and the Department of Proteomics and Signal Transduction at Max-Planck Institute of Biochemistry for useful comments and support.

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Correspondence to Shao-En Ong.

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Ong, SE., Mann, M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc 1, 2650–2660 (2006). https://doi.org/10.1038/nprot.2006.427

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