Summary
Quantitative proteomics has become a pivotal tool that has been applied to the investigation of many different biological processes such diverse as the detection of biomarkers in tissue samples, the regulation of cell signaling, and the characterization of protein interactions. Stable isotope labeling techniques have facilitated the precise quantitation of changes in protein abundance by mass spectrometry. Among different choices, Stable Isotope Labeling by Amino acids in Cell culture (SILAC) is an easy and reliable method for unbiased comparative proteomic experiments, which has been employed to study post-translational modifications such as protein phosphorylation and methylation, to characterize signaling pathways and to determine specific protein interactions. Here we describe detailed procedures for SILAC experiments in mammalian and yeast cells.
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References
Blagoev, B., Kratchmarova, I., Ong, S.E., Nielsen, M., Foster, L.J. and Mann, M. (2003) A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat Biotechnol. 21(3), 315–318.
Gruhler, A., Olsen, J.V., Shabaz, M., Mortensen, P., Faergman, N., Mann, M. and Jensen, O.N. (2005) Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. Molecular and Cellular Proteomics. 4(3), 310–27.
Ranish, J.A., Yi, E.C., Leslie, D.M., Purvine, S.O., Goodlett, D.R., Eng, J. and Aebersold, R. (2003) The study of macromolecular complexes by quantitative proteomics. Nat Genet. 33(3), 349–55.
Schulze, W. and Mann, M. (2004) A novel proteomic screen for peptide-protein interactions. J Biol Chem. 279(11), 10756–10764.
Dunkley, T.P., Watson, R., Griffin, J.L., Dupree, P. and Lilley, K.S. (2004) Localization of organelle proteins by isotope tagging (LOPIT). Mol Cell Proteomics. 3(11), 1128–34.
Andersen, J.S., Lam, Y.W., Leung, A.K., Ong, S.E., Lyon, C.E., Lamond, A.I. and Mann, M. (2005) Nucleolar proteome dynamics. Nature. 433(7021), 77–83.
Westermeier, R. and Marouga, R. (2005) Protein detection methods in proteomics research. Biosci Rep. 25(1–2), 19–32.
Ong, S.E. and Mann, M. (2005) Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol. 1(5), 252–62.
Leitner, A. and Lindner, W. (2004) Current chemical tagging strategies for proteome analysis by mass spectrometry. J Chromatog B. 813(1–2), 1.
Krijgsveld, J., Ketting, R.F., Mahmoudi, T., Johansen, J., Artal-Sanz, M., Verrijzer, C.P., Plasterk, R.H. and Heck, A.J. (2003) Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics. Nat Biotechnol. 21(8), 927–31.
Lahm, H.W. and Langen, H. (2000) Mass spectrometry: a tool for the identification of proteins separated by gels. Electrophoresis. 21(11), 2105–14.
Oda, Y., Huang, K., Cross, F.R., Cowburn, D. and Chait, B.T. (1999) Accurate quantitation of protein expression and site-specific phosphorylation. PNAS. 96(12), 6591–6596.
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(5), 376–86.
Zhu, H., Pan, S., Gu, S., Bradbury, E.M. and Chen, X. (2002) Amino acid residue specific stable isotope labeling for quantiative proteomics. Rapid Communications in Mass Spectrometry. 16(22), 2115–2123.
Gruhler, A., Schulze, W.X., Matthiesen, R., Mann, M. and Jensen, O.N. (2005) Stable Isotope Labeling of Arabidopsis thaliana Cells and Quantitative Proteomics by Mass Spectrometry. Mol Cell Proteomics. 4(11), 1697–1709.
Blagoev, B., Ong, S.E., Kratchmarova, I. and Mann, M. (2004) Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat Biotechnol. 22(9), 1139–45.
Kratchmarova, I., Blagoev, B., Haack-Sorensen, M., Kassem, M. and Mann, M. (2005) Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation. Science. 308(5727), 1472–7.
Olsen, J.V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P. and Mann, M. (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell, 127, 635–648.
Amanchy, R., Kalume, D.E., Iwahori, A., Zhong, J. and Pandey, A. (2005) Phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC). J Proteome Res. 4(5), 1661–71.
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(2), 173–81.
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Gruhler, s., Kratchmarova, I. (2008). Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). In: Posch, A. (eds) 2D PAGE: Sample Preparation and Fractionation. Methods in Molecular Biology™, vol 424. Humana Press. https://doi.org/10.1007/978-1-60327-064-9_9
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DOI: https://doi.org/10.1007/978-1-60327-064-9_9
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