Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/SILAC

right|thumb|420px|The principle of SILAC. Cells differentially labeled by growing them in light medium with normal arginine (Arg-0, grey colour) or medium with heavy arginine (Arg-6, red colour). Metabolic incorporation of the amino acids into the proteins results in a mass shift of the corresponding peptides. This mass shift can be detected by a mass spectrometer as indicated by the depicted mass spectra. When both samples are combined, the ratio of peak intensities in the mass spectrum reflects the relative protein abundance. In this example, the labeled protein has the same abundance in both samples (ratio 1). SILAC (stable isotope labeling by amino acids in cell culture) is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labeling.Jiang H, English, AM 2002 Quantitative Analysis of the Yeast Proteome by Incorporation of Isotopically Labeled Leucine Journal of Proteome Research 1 345–50 12645890 http://pubs.acs.org/doi/abs/10.1021/pr025523f 10.1021/pr025523f 4Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M 2002 Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics Molecular & Cellular Proteomics 1 376–86 12118079 http://www.mcponline.org/cgi/content/abstract/1/5/376 10.1074/mcp.M200025-MCP200 5Zhu H, Pan S, Gu S, Bradbury EM, Chen X 2002 Amino acid residue specific stable isotope labeling for quantitative proteomics Rapid Communications in Mass Spectrometry 16 2115–23 12415544 http://www3.interscience.wiley.com/journal/99018948/abstract?CRETRY=1&SRETRY=0 10.1002/rcm.831 22 It is a popular method for quantitative proteomics. Procedure Two populations of cells are cultivated in cell culture. One of the cell populations is fed with growth medium containing normal amino acids. In contrast, the second population is fed with growth medium containing amino acids labeled with stable (non-radioactive) heavy isotopes. For example, the medium can contain arginine labeled with six carbon-13 atoms (13C) instead of the normal carbon-12 (12C). When the cells are growing in this medium, they incorporate the heavy arginine into all of their proteins. Therefore, all of the arginine containing peptides are now 6 Da heavier than their normal counterparts. The trick is that the proteins from both cell populations can be combined and analyzed together by mass spectrometry. Pairs of chemically identical peptides of different stable-isotope composition can be differentiated in a mass spectrometer owing to their mass difference. The ratio of peak intensities in the mass spectrum for such peptide pairs reflects the abundance ratio for the two proteins. Applications A SILAC approach involving incorporation of tyrosine labeled with nine carbon-13 atoms (13C) instead of the normal carbon-12 (12C) has been utilized to study tyrosine kinase substrates in signaling pathways.Ibarrola N, Molina H, Iwahori A, Pandey A 2004 A novel proteomic approach for specific identification of tyrosine kinase substrates using 13C tyrosine The Journal of Biological Chemistry 279 15805–13 14739304 http://www.jbc.org/content/279/16/15805.long 10.1074/jbc.M311714200 16 SILAC has emerged as a very powerful method to study cell signaling, post translation modifications such as phosphorylation, Ibarrola N, Kalume DE, Gronborg M, Iwahori A, Pandey A 2003 A Proteomic Approach for Quantitation of Phosphorylation Using Stable Isotope Labeling in Cell Culture Analytical Chemistry 75 6043–49 14615979 http://pubs.acs.org/doi/abs/10.1021/ac034931f 10.1021/ac034931f 22 protein-protein interaction and regulation of gene expression. Standardized protocols of SILAC for various application have also been published.Amanchy R, Kalume DE, Pandey A 2005 Stable isotope labeling with amino acids in cell culture (SILAC) for studying dynamics of protein abundance and posttranslational modifications Science's STKE 2005 pl2 14739304 http://stke.sciencemag.org/cgi/content/full/sigtrans;2005/267/pl2 10.1126/stke.2672005pl2 16 Harsha HC, Molina H, Pandey A 2008 Quantitative proteomics using stable isotope labeling with amino acids in cell culture Nature Protocols 3 505–16 18323819 http://www.nature.com/nprot/journal/v3/n3/abs/nprot.2008.2.html 10.1038/nprot.2008.2 3 Pulsed SILAC Pulsed SILAC (pSILAC) is a variation of the SILAC method where the labelled amino acids are added to the growth medium for only a short period of time. This allows monitoring differences in de novo protein production rather than raw concentration. 10.1002/pmic.200800275 9 1 205–9 Schwanhäusser Björn Manfred Gossen, Gunnar Dittmar, Matthias Selbach Global analysis of cellular protein translation by pulsed SILAC Proteomics 2009-03-17 2009-01 19053139 http://www3.interscience.wiley.com/journal/121543086/abstract See also ICAT Isobaric labeling Tandem mass tags (TMT) Isobaric tags for relative and absolute quantitation (iTRAQ) Label-free quantification References Bibliography Ong SE, Kratchmarova I, Mann M 2003 Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC) Journal of Proteome Research 2 173–81 12716131 http://pubs.acs.org/cgi-bin/abstract.cgi/jprobs/2003/2/i02/abs/pr0255708.html 10.1021/pr0255708 2 Ong SE, Mann M 2006 A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC) Nature Protocols 1 2650–60 17406521 http://www.nature.com/nprot/journal/v1/n6/abs/nprot.2006.427.html 10.1038/nprot.2006.427 6 Ong SE, Mann M 2007 Stable isotope labeling by amino acids in cell culture for quantitative proteomics Methods in Molecular Biology 359 37–52 17484109 10.1007/978-1-59745-255-7_3 Notes External links SILAC resource Mann Lab SILAC resource Pandey Lab SILAC resource Center for Experimental Bioinformatics (CEBI) Category:Biochemistry methods Category:Biotechnology Category:Mass spectrometry Category:Proteomics Category:Protein-protein interaction assays