Proteomics

SILAC

Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)

Akhilesh Pandey, PhD
Johns Hopkins University


  • Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)
  • NeuCode™ SILAC

SILAC refers to labeling cultured cells with heavy amino acids for quantitative proteomic analysis. Labeling an entire proteome with heavy amino acids in vivo generates an ideal standard for quantitative proteomics. When a heavy labeled proteome is mixed with an unlabeled proteome then digested, every unlabeled peptide identified by the mass spectrometer can be quantified by its corresponding heavy peptide. In SILAC, the tryptic amino acids, arginine(R) and lysine (K), contain heavy stable isotopes, so if digesting with trypsin, every peptide is labeled. This metabolic labeling strategy has been employed by hundreds of proteomic studies (see example references below). The advantage of metabolic labeling over in vitro tagging techniques is that the heavy and unlabeled samples are mixed before sample preparation, preventing variability between preparations distorting the quantitation results.  This is especially important when extensive sample preparation (e.g. isolation of an organelle) is required. 

 

 

Frequently Asked Questions 

Does CIL offer media, kits and reagents for SILAC?

SILAC Protein Quantitation Kits

How many samples can be tested with the SILAC Protein Quantitation Kit?

Each kit contains 500 mL media, which can isotopically label approximately 1 x 1012 cells, depending on cell type.

Do you have a protocol?

Yes, click here to download the SILAC Protein Quantitation Kit instructions for use.

Would 0.8 mM of Lysine or Arginine preloaded resins provide approximately 8 synthesis at 0.1 mM scale?

Yes, theoretically 0.8 mM of resin would provide 8 synthesis at 0.1MM scale. Final yields are always dependent on actual sequence and the efficiency of the couplings.

 

References 

Ma,Y; McClatchy, D.B.; Barkallah, S.; Wood, W.W.; Yates, Lii JR.2017. HILAQ: A Novel Strategry for Newly Synthesized Protein Quantification. J Proteome Res,[Epub ahead of print]. PMID: 28437088

Singh, S.A.; Andraski, A.B.; Pieper, B. 2016. Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring. J Lipid Res., 57(4):714-28. PMID: 26862155

McClatchy, D.B.; Ma,Y; Liu, C.;Stein, B.D.; Martínez-Bartolomé, S.; Vasquez, D.; Hellberg, K.; Shaw, R.J.; Yates, J.R. 3rd. 2015. Pulsed Azidohomoalanine Labeling in Mammals (PALM) Detects Changes in Liver-Specific LKB1 Knockout Mice. J Proteome Res, 14(11):4815-22. PMID: 26445171

Cox, T.R.; Schoof, E.M.; Gartland, A. 2015. Dataset for the proteomic inventory and quantitative analysis of the breast cancer hypoxic secretome associated with osteotropism. Data Brief, 5:621-5. PMID: 26649326

McShane, A.J.; Bajrami, B.; Ramos, A.A. 2014. Targeted proteomic quantitation of the absolute expression and turnover of cystic fibrosis transmembrane conductance regulator in the apical plasma membrane. J Proteome Res., 13(11):4676-85. PMID: 25227318

Bagert, J.D.; Xie, Y.J.; Sweredoski, M.J. 2014. Quantitative, time-resolved proteomic analysis by combining bioorthogonal noncanonical amino acid tagging and pulsed stable isotope labeling by amino acids in cell culture. Mol Cell Proteomics, 13(5):1352-8. PMID: 24563536

Edfors, F.; Bostrom, T.; Forsstrom, B. 2014. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins. Mol Cell Proteomics, 13(6):1611-24. PMID: 24722731

Rayavarapu, S.; Coley, W.; Cakir, E. 2013. Identification of disease specific pathways using in vivo SILAC proteomics in dystrophin deficient mdx mouse. Mol Cell Proteomics, 12(5):1061-73. PMID: 23297347

Kristensen, A.R.; Gsponer, J.; Foster, L.J. 2013. Protein synthesis rate is the predominant regulator of protein expression during differentiation. Mol Syst Biol., 9:689. PMID: 24045637

Liao, L.; McClatchy, D.B.; Park, S.K.; Xu, T.; Lu, B.; Yates III, J.R. 2008. Quantitative analysis of brain nuclear phosphoproteins identifies developmentally regulated phosphorylation events. J Proteome Res, 7(11):4743-55. PMID: 18823140

Selbach, M.; Mann, M. 2006. Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nature Methods, 3, 981-983. PMID:17072306

Mann, M. 2006. Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol, 7, 952-958. PMID: 17139335

Amanchy, R.; Kalume. D.E.; Pandey, A. 2005. Stable isotope labeling with amino acids in cell culture (SILAC) for studying dynamics of protein abundance and posttranslational modifications. Sci STKE, 267, 1-20. PMID:15657263

Kratchmarova, I.; Blagoev. B.; Haack-Sorensen. M.; Kassem, M.; Mann, M. 2005. Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation. Science, 308, 1472-1477. PMID:15933201

Blagoev, B.; Ong, S.E.; Kratchmarova. I.; Mann. M. 2004. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nature Biotechnology, 22, 1139-1145. PMID:15314609

Ong, S.E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D.B.; 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. Mol Cell Proteomics, 1, 376-386. PMID:12118079

Kevin Millis, PhD

Kevin Millis, PhD

Senior Scientist, Application Development Manager

Kevin Millis, PhD, is the Senior Scientist and Market Development Manager for all NMR and Mass Spectrometry product lines. Kevin is responsible for Technical Services both internally and externally for all CIL customers as well as being responsible for the application and market development for the CIL products.

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Andrew Percy, PhD

Andrew Percy, PhD

Senior Applications Chemist - Mass Spectrometry

Dr. Andrew Percy is the Senior Applications Chemist for Mass Spectrometry. His responsibilities minimally involve overseeing product development, identifying new product market opportunities, assisting in the analysis of products for MS ‘omics applications, and providing technical support to customers.

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