Stable isotope labeling by amino acids in cell culture

The principle of SILAC. Cells are differentially labeled by growing them in light medium with normal arginine (Arg-0, blue color) or medium with heavy arginine (Arg-6, red color). 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/with 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.[1][2][3][4] 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. Thereafter, all peptides containing a single arginine are 6 Da heavier than their normal counterparts. Alternatively, uniform labeling with 13C or 15N can be used. 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.[5] SILAC has emerged as a very powerful method to study cell signaling, post translation modifications such as phosphorylation,[5][6] protein–protein interaction and regulation of gene expression. In addition, SILAC has become an important method in secretomics, the global study of secreted proteins and secretory pathways.[7] It can be used to distinguish between proteins secreted by cells in culture and serum contaminants.[8] Standardized protocols of SILAC for various application have also been published.[9][10]

While SILAC had been mostly used in studying eukaryotic cells and cell cultures, it had been recently employed in bacteria and its multicellular biofilm in antibiotic tolerance, to differentiate tolerance and sensitive subpopulations.[11]

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.[12]

It had also been used to study biofilm tolerance to antibiotics to differentiate tolerant and sensitive subpopulations [11]

References

  1. ↑ Oda Y, Huang K, Cross FR, Cowburn D, Chait BT (1999). "Accurate quantitation of protein expression and site-specific phosphorylation.". Proc Natl Acad Sci U S A. 96 (12): 6591–6. PMC 21959 Freely accessible. PMID 10359756. doi:10.1073/pnas.96.12.6591.
  2. ↑ Jiang H, English, AM (2002). "Quantitative Analysis of the Yeast Proteome by Incorporation of Isotopically Labeled Leucine". Journal of Proteome Research. 1 (4): 345–50. PMID 12645890. doi:10.1021/pr025523f.
  3. ↑ Ong 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 (5): 376–86. PMID 12118079. doi:10.1074/mcp.M200025-MCP200.
  4. ↑ Zhu 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 (22): 2115–23. PMID 12415544. doi:10.1002/rcm.831.
  5. 1 2 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 (16): 15805–13. PMID 14739304. doi:10.1074/jbc.M311714200.
  6. ↑ 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 (22): 6043–49. PMID 14615979. doi:10.1021/ac034931f.
  7. ↑ Hathout, Yetrib (2007). "Approaches to the study of the cell secretome". Expert Review of Proteomics. 4 (2): 239–48. PMID 17425459. doi:10.1586/14789450.4.2.239.
  8. ↑ Polacek, Martin; Bruun, Jack-Ansgar; Johansen, Oddmund; Martinez, Inigo (2010). "Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology". Journal of Orthopaedic Research. 28 (8): 1040–9. PMID 20108312. doi:10.1002/jor.21067.
  9. ↑ 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 (16): pl2. PMID 15657263. doi:10.1126/stke.2672005pl2.
  10. ↑ Harsha HC, Molina H, Pandey A (2008). "Quantitative proteomics using stable isotope labeling with amino acids in cell culture". Nature Protocols. 3 (3): 505–16. PMID 18323819. doi:10.1038/nprot.2008.2.
  11. 1 2 Chua SL, Yam JK, Sze KS, Yang L (2016). "Selective labelling and eradication of antibiotic-tolerant bacterial populations in Pseudomonas aeruginosa biofilms". Nat Commun. 7: 10750. PMC 4762895 Freely accessible. PMID 26892159. doi:10.1038/ncomms10750.
  12. ↑ Schwanhäusser, Björn; Manfred Gossen; Gunnar Dittmar; Matthias Selbach (January 2009). "Global analysis of cellular protein translation by pulsed SILAC". Proteomics. 9 (1): 205–9. PMID 19053139. doi:10.1002/pmic.200800275. Retrieved 2009-03-17.

Further reading

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