SILAC

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Schematic sequence of the SILAC. In this example the amounts of the peptide under consideration are approximately the same.

SILAC (abbreviation for stable isotope labeling by / with amino acids in cell culture ) is a mass spectrometric method for determining quantities using isotope labeling . SILAC is used in proteomics .

principle

Two originally identical cell cultures are cultivated with different nutrient media . One of the cell cultures only receives amino acids in the medium that carry a heavy isotope. For example, the medium can contain arginine with six 13 carbon atoms instead of the usual 12 C. During protein synthesis , the proteins are marked. As a result, all arginine-containing peptides are six daltons heavier than the non-labeled peptides of the other cell culture. Alternatively, the two cell cultures can also be labeled with 13 C or 15 N. For the analysis, the proteins from both cell cultures are combined and measured together. The marked peptides can be identified on the basis of the different masses. The ratios of the signal intensities correspond to the quantitative ratios of the peptides.

Alternatives to SILAC are e.g. B. ICAT that Isobarenmarkierung that Tandem Mass Tags (TMT), iTRAQ and label-free quantification ( English label free quantification ).

Applications

A SILAC-based approach has been used to study signal transduction , e.g. B. to determine the substrates of receptor tyrosine kinases , post-translational modifications such as phosphorylations , protein-protein interaction and to investigate the regulation of gene expression .

Pulsed SILAC

Pulsed SILAC (pSILAC) is a variant of SILAC in which the marked amino acids are only added to the cell cultures for a limited time, whereby newly formed proteins are measured and their synthesis rate is derived.

literature

  • Ong SE, Kratchmarova I, Mann M: Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC) . In: Journal of Proteome Research . 2, No. 2, 2003, pp. 173-81. doi : 10.1021 / pr0255708 . PMID 12716131 .
  • Ong SE, Mann M: A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC) . In: Nature Protocols . 1, No. 6, 2006, pp. 2650-60. doi : 10.1038 / nprot.2006.427 . PMID 17406521 .
  • Ong SE, Mann M: Stable isotope labeling by amino acids in cell culture for quantitative proteomics . In: Methods in Molecular Biology . 359, 2007, pp. 37-52. doi : 10.1007 / 978-1-59745-255-7_3 . PMID 17484109 .

Web links

Individual evidence

  1. ↑ Oda Y, Huang K, Cross FR, Cowburn D, Chait BT: Accurate quantitation of protein expression and site-specific phosphorylation. . In: Proc Natl Acad Sci USA . 96, No. 12, 1999, pp. 6591-6. PMID 10359756 . PMC 21959 (free full text).
  2. ↑ Jiang H, English, AM: Quantitative Analysis of the Yeast Proteome by Incorporation of Isotopically Labeled Leucine . In: Journal of Proteome Research . 1, No. 4, 2002, pp. 345-50. doi : 10.1021 / pr025523f . PMID 12645890 .
  3. ↑ Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M: Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics . In: Molecular & Cellular Proteomics . 1, No. 5, 2002, pp. 376-86. doi : 10.1074 / mcp.M200025-MCP200 . PMID 12118079 .
  4. ↑ Zhu H, Pan S, Gu S, Bradbury EM, Chen X: Amino acid residue specific stable isotope labeling for quantitative proteomics . In: Rapid Communications in Mass Spectrometry . 16, No. 22, 2002, pp. 2115-23. doi : 10.1002 / rcm.831 . PMID 12415544 .
  5. ↑ Amanchy R, Kalume DE, Pandey A: Stable isotope labeling with amino acids in cell culture (SILAC) for studying dynamics of protein abundance and posttranslational modifications . In: Science's STKE . 2005, No. 16, 2005, p. Pl2. doi : 10.1126 / stke.2672005pl2 . PMID 15657263 .
  6. ↑ Harsha HC, Molina H, Pandey A: Quantitative proteomics using stable isotope labeling with amino acids in cell culture . In: Nature Protocols . 3, No. 3, 2008, pp. 505-16. doi : 10.1038 / nprot.2008.2 . PMID 18323819 .
  7. ↑ a b Ibarrola N, Molina H, Iwahori A, Pandey A: A novel proteomic approach for specific identification of tyrosine kinase substrates using 13C tyrosine . In: The Journal of Biological Chemistry . 279, No. 16, 2004, pp. 15805-13. doi : 10.1074 / jbc.M311714200 . PMID 14739304 .
  8. ↑ Ibarrola N, Kalume DE, Gronborg M, Iwahori A, Pandey A: A Proteomic Approach for Quantitation of Phosphorylation Using Stable Isotope Labeling in Cell Culture . In: Analytical Chemistry . 75, No. 22, 2003, pp. 6043-49. doi : 10.1021 / ac034931f . PMID 14615979 .
  9. ↑ Yetrib Hathout: Approaches to the study of the cell secretome . In: Expert Review of Proteomics . 4, No. 2, 2007, pp. 239-48. doi : 10.1586 / 14789450.4.2.239 . PMID 17425459 .
  10. ↑ Martin Polacek, Jack-Ansgar Bruun, Oddmund Johansen, Inigo Martinez: Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology . In: Journal of Orthopedic Research . 28, No. 8, 2010, pp. 1040-9. doi : 10.1002 / jor.21067 . PMID 20108312 .
  11. ↑ Björn Schwanhäusser, Manfred Gossen, Gunnar Dittmar, Matthias Selbach: Global analysis of cellular protein translation by pulsed SILAC . In: Proteomics . 9, No. 1, September, pp. 205-9. doi : 10.1002 / pmic.200800275 . PMID 19053139 .