Stable isotope labeling by amino acids in cell culture explained

Stable isotope labeling by/with amino acids in cell culture (SILAC) 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. Proteins from both cell populations are combined and analyzed together by mass spectrometry as 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.^{[5] [3]}

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.^[6] SILAC has emerged as a very powerful method to study cell signaling, post translation modifications such as phosphorylation,^{[6] [7]} 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.^[8] It can be used to distinguish between proteins secreted by cells in culture and serum contaminants.^[9] Standardized protocols of SILAC for various applications have also been published.^{[10] [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]

NeuCode SILAC

Traditionally the level of multiplexing in SILAC was limited due to the number of SILAC isotopes available. Recently, a new technique called NeuCode (neutron encoding) SILAC, has augmented the level of multiplexing achievable with metabolic labeling (up to 4).^[13] The NeuCode amino acid method is similar to SILAC but differs in that the labeling only utilizes heavy amino acids. The use of only heavy amino acids eliminates the need for 100% incorporation of amino acids needed for SILAC. The increased multiplexing capability of NeuCode amino acids is from the use of mass defects from extra neutrons in the stable isotopes. These small mass differences however need to be resolved on high-resolution mass spectrometers.

Further reading

• Ong SE, Kratchmarova I, Mann M . Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC) . Journal of Proteome Research . 2 . 2 . 173–181 . 2003 . 12716131 . 10.1021/pr0255708 .
• Ong SE, Mann M . A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC) . Nature Protocols . 1 . 6 . 2650–2660 . 2006 . 17406521 . 10.1038/nprot.2006.427 . 10651610 .
• Book: Ong SE, Mann M . Quantitative Proteomics by Mass Spectrometry . 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. 978-1-58829-571-2 .

External links

• SILAC resource Mann Lab
• SILAC resource Pandey Lab
• SILAC resource Center for Experimental Bioinformatics (CEBI)

Notes and References

1. Oda Y, Huang K, Cross FR, Cowburn D, Chait BT . Accurate quantitation of protein expression and site-specific phosphorylation . Proceedings of the National Academy of Sciences of the United States of America . 96 . 12 . 6591–6596 . June 1999 . 10359756 . 21959 . 10.1073/pnas.96.12.6591 . free . 1999PNAS...96.6591O .
2. Jiang H, English AM . Quantitative analysis of the yeast proteome by incorporation of isotopically labeled leucine . Journal of Proteome Research . 1 . 4 . 345–350 . 2002 . 12645890 . 10.1021/pr025523f .
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 . Molecular & Cellular Proteomics . 1 . 5 . 376–386 . May 2002 . 12118079 . 10.1074/mcp.M200025-MCP200 . free .
4. Zhu H, Pan S, Gu S, Bradbury EM, Chen X . Amino acid residue specific stable isotope labeling for quantitative proteomics . Rapid Communications in Mass Spectrometry . 16 . 22 . 2115–2123 . 2002 . 12415544 . 10.1002/rcm.831 . 2002RCMS...16.2115Z .
5. Schoeters F, Van Dijck P . Protein-Protein Interactions in Candida albicans . Frontiers in Microbiology . 10 . 1792 . 2019 . 31440220 . 6693483 . 10.3389/fmicb.2019.01792 . free .
6. Ibarrola N, Molina H, Iwahori A, Pandey A . A novel proteomic approach for specific identification of tyrosine kinase substrates using [13C]tyrosine . The Journal of Biological Chemistry . 279 . 16 . 15805–15813 . April 2004 . 14739304 . 10.1074/jbc.M311714200 . free .
7. Ibarrola N, Kalume DE, Gronborg M, Iwahori A, Pandey A . A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture . Analytical Chemistry . 75 . 22 . 6043–6049 . November 2003 . 14615979 . 10.1021/ac034931f .
8. Hathout Y . Approaches to the study of the cell secretome . Expert Review of Proteomics . 4 . 2 . 239–248 . April 2007 . 17425459 . 10.1586/14789450.4.2.239 . 26169223 .
9. Polacek M, Bruun JA, Johansen O, Martinez I . Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology . Journal of Orthopaedic Research . 28 . 8 . 1040–1049 . August 2010 . 20108312 . 10.1002/jor.21067 . 41057768 . free .
10. 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 . Science's STKE . 2005 . 267 . pl2 . January 2005 . 15657263 . 10.1126/stke.2672005pl2 . 12089034 .
11. Harsha HC, Molina H, Pandey A . Quantitative proteomics using stable isotope labeling with amino acids in cell culture . Nature Protocols . 3 . 3 . 505–516 . 2008 . 18323819 . 10.1038/nprot.2008.2 . 24190501 .
12. Schwanhäusser B, Gossen M, Dittmar G, Selbach M . Global analysis of cellular protein translation by pulsed SILAC . Proteomics . 9 . 1 . 205–209 . January 2009 . 19053139 . 10.1002/pmic.200800275 . 23130202 .
13. Merrill AE, Hebert AS, MacGilvray ME, Rose CM, Bailey DJ, Bradley JC, Wood WW, El Masri M, Westphall MS, Gasch AP, Coon JJ . 6 . NeuCode labels for relative protein quantification . Molecular & Cellular Proteomics . 13 . 9 . 2503–2512 . September 2014 . 24938287 . 4159665 . 10.1074/mcp.M114.040287 . free .