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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)
  • SILAC Kits and Reagents
  • SILAC Media Compositions
  • 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 from distorting the final quantitation results. This is especially important when extensive sample preparation (e.g. isolation of an organelle) is required. 

Resources 

SILAC Kits and Reagents
L-Azidohomoalanine·HCl (AHA) 

 

Note: NeuCode™ amino acids are supplied by Thermo Fisher Scientific (for SILAC applications), while CIL offers the Mouse Express® NeuCode™ mouse feed (for SILAM studies). NeuCode™ is a trademark of the Wisconsin Alumni Research Foundation (WARF), while Mouse Express® is a registered trademark of CIL.
 
Please visit the NeuCode™ section of Thermo’s SILAC Metabolic Labeling Systems webpage for more information on the NeuCode™ amino acids.
 
 
 

Frequently Asked Questions 

What is the typical labeling period required for near 100% isotope incorporation into the cell proteome?

The number of cell doublings required for near complete heavy isotope incorporation is typically 5-6. This has been demonstrated with numerous cell lines (e.g., HeLa, HEK293).

How does one prevent L-Arg to L-Pro conversion in SILAC-MS experiments?

There are procedural measures that should be undertaken to help minimize this adverse metabolic reaction. For design notes and tips/tricks on best practices for executing SILAC-MS experiments, please refer to Mann et al.’s Nature Protocols article (PMID: 17406521).

Which stable isotope-labeled reagents are typically utilized in a triple (or triplex) SILAC experiment?

A triple SILAC-MS experiment generally utilizes L-Lys and L-Arg in the following three isotope labeling states:

Labeling Type Typical Compounds and Catalog Numbers
Light (L) L-Arg (unlabeled – ULM-8347) and L-Lys (unlabeled – ULM-8766)
Medium (M) 13C6 L-Arg (CLM-2265) and D4 L-Lys (DLM-2640)
Heavy (H) 13C6, 15N4 L-Arg (CNLM-539-H) and 13C6, 15N2 L-Lys (CNLM-291-H)

 

 

 


 

 

References 

Shin, J.; Rhim, J.; Kown, Y.; et al. 2019. Comparative analysis of differentially secreted proteins in serum-free and serum-containing media by using BONCAT and pulsed SILAC. Sci Rep, 9(1), 3096. PMID: 30816242

Duan, Q.; Li, D.; Xiong, L.; et al. 2019. SILAC quantitative proteomics and biochemical analyses reveal a novel molecular mechanism by which ADAM12S promotes the proliferation, migration, and invasion of small cell lung cancer cells through upregulating hexokinase 1. J Proteome Res, 18(7), 2903-2914. PMID: 31117637

Han, J.; Yi, S.; Zhao, X.; et al. 2019. Improved SILAC method for double labeling of bacterial proteome. J Proteomics, 194, 89-98. PMID: 30553074

McMillan, L.J.; Hwang, S.; Farah, R.E.; et al. 2018. Multiplex quantitative SILAC for analysis of archaeal proteomes: a case study of oxidative stress responses. Environ Microbiol, 20(1), 385-401. PMID: 29194950

Heo, S.; Diering, G.H.; Na, C.H.; et al. 2018. Identification of long-lived synaptic proteins by proteomic analysis of synaptosome protein turnover. Proc Natl Acad Sci U S A, 115(16), E3827-E3836. PMID: 29610302

Wang, Q.; Guo, L.; Strawser, C.J.; et al. 2018. Low apolipoprotein A-I levels in Friedreich's ataxia and in frataxin-deficient cells: implications for therapy. PLoS One, 13(2), e0192779. PMID: 29447225

Overmyer, K.A.; Tyanova, S.; Herbert, A.S.; et al. 2018. Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice. Nat Protoc, 3(1), 293-306. PMID: 29323663

Peikert, C.D.; Mani, J.; Morgenstern, M.; et al. 2017. Charting organellar importomes by quantitative mass spectrometry. Nat Commun, 8, 15272. PMID: 28485388

Ma, Y.; McClatchy, D.B.; Barkallah, S.; et al. 2017. HILAQ: a novel strategry for newly synthesized protein quantification. J Proteome Res, 16(6), 2213-2220. PMID: 28437088
 
Gong, J.; Körner, R.; Gaitanos, L.; et al. 2016. Exosomes mediate cell contact-independent ephrin-Eph signaling during axon guidance. J Cell Biol, 214(1), 35-44. PMID: 27354374
 
Gonneaud, A.; Jones, C.; Turgeon, N.; et al. 2016. A SILAC-based method for quantitative proteomic analysis of intestinal organoids. Sci Rep, 6, 38195. PMID: 27901089
 
Singh, S.A.; Andraski, A.B.; Pieper, B.; et al. 2016. Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring. J Lipid Res, 57(4), 714-728. PMID: 26862155
 
Bagert, J.D.; Xie, Y.J.; Sweredoski, M.J.; et al. 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), 1352-1358. PMID: 24563536
 
Ong, S.E.; Mann, M. 2006. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc, 1(6), 2650-2660. PMID: 17406521
 
Selbach, M.; Mann, M. 2006. Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nat Methods, 3(12), 981-983. PMID: 17072306
 
Mann, M. 2006. Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol, 7(12), 952-958. PMID: 17139335
 
Ong, S.E.; Blagoev, B.; Kratchmarova, I.; et al. 2002. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics, 1(5), 376-386. PMID: 12118079

 

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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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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