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Quantitative proteomics using stable isotope labeling with amino acids in cell culture


Stable isotope labeling with amino acids in cell culture (SILAC) is a simple in vivo labeling strategy for mass spectrometry-based quantitative proteomics. It relies on the metabolic incorporation of nonradioactive heavy isotopic forms of amino acids into cellular proteins, which can be readily distinguished in a mass spectrometer. As the samples are mixed before processing in the SILAC methodology, the sample handling errors are also minimized. Here we present protocols for using SILAC in the following types of experiments: (i) studying inducible protein complexes, (ii) identification of Tyr kinase substrates, (iii) differential membrane proteomics and (iv) studying temporal dynamics using SILAC 5-plexing. Although the overall time is largely dependent on the rate of cell growth and various sample processing steps employed, a typical SILAC experiment from start to finish, including data analysis, should take anywhere between 20 and 25 d.

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Figure 1: Workflow of a stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic experiment.
Figure 2: Incorporation of heavy Lys (13C6) in the presence of complete serum.
Figure 3: Use of 5-plex stable isotope labeling with amino acids in cell culture experiment to monitor protein dynamics.
Figure 4: Liquid chromatography (LC) and mass spectrometry (MS) setup.
Figure 5: Distinguishing nonspecific and specific interactions using stable isotope labeling with amino acids in cell culture.
Figure 6: Mass resolution issues in ion trap mass spectrometers.

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  1. Ong, S.E. et al. 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 (2002).

    Article  CAS  Google Scholar 

  2. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    Article  CAS  Google Scholar 

  3. Ross, P.L. et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell Proteomics 3, 1159–1169 (2004).

    Google Scholar 

  4. Schnolzer, M., Jedrzejewski, P. & Lehmann, W.D. Protease-catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry. Electrophoresis 17, 945–953 (1996).

    Article  CAS  Google Scholar 

  5. Zhang, R., Sioma, C.S., Thompson, R.A., Xiong, L. & Regnier, F.E. Controlling deuterium isotope effects in comparative proteomics. Anal. Chem. 74, 3662–3669 (2002).

    Article  CAS  Google Scholar 

  6. Olsen, J.V. et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648 (2006).

    Article  CAS  Google Scholar 

  7. Blagoev, B., Ong, S.E., Kratchmarova, I. & Mann, M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat. Biotechnol. 22, 1139–1145 (2004).

    Article  CAS  Google Scholar 

  8. Neher, S.B. et al. Proteomic profiling of ClpXP substrates after DNA damage reveals extensive instability within SOS regulon. Mol. Cell 22, 193–204 (2006).

    Article  CAS  Google Scholar 

  9. Gronborg, M. et al. Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach. Mol. Cell Proteomics 5, 157–171 (2005).

    Article  Google Scholar 

  10. Guerrero, C., Tagwerker, C., Kaiser, P. & Huang, L. An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network. Mol. Cell Proteomics 5, 366–378 (2006).

    Article  CAS  Google Scholar 

  11. Ong, S.E., Kratchmarova, I. & Mann, M. Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC). J. Proteome Res. 2, 173–181 (2003).

    Article  CAS  Google Scholar 

  12. Villen, J., Beausoleil, S.A., Gerber, S.A. & Gygi, S.P. Large-scale phosphorylation analysis of mouse liver. Proc. Natl. Acad. Sci. USA 104, 1488–1493 (2007).

    Article  CAS  Google Scholar 

  13. Rush, J. et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol. 23, 94–101 (2005).

    Article  CAS  Google Scholar 

  14. Anderson, L. & Porath, J. Isolation of phosphoproteins by immobilized. Metal (Fe3+) affinity chromatography. Anal. Biochem. 154, 250–254 (1986).

    Article  Google Scholar 

  15. Stensballe, A., Andersen, S. & Jensen, O.N. Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis. Proteomics 1, 207–222 (2001).

    Article  CAS  Google Scholar 

  16. Larsen, M.R., Thingholm, T.E., Jensen, O.N., Roepstorff, P. & Jorgensen, T. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell Proteomics 4, 873–886 (2005).

    Article  CAS  Google Scholar 

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This work was supported by National Institutes of Health Roadmap grant 'Technology Center for Networks and Pathways' (RR020839).

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Correspondence to Akhilesh Pandey.

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Harsha, H., Molina, H. & Pandey, A. Quantitative proteomics using stable isotope labeling with amino acids in cell culture. Nat Protoc 3, 505–516 (2008).

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