Skip to main content

Design and Application of Super-SILAC for Proteome Quantification

  • Protocol
  • First Online:
Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1188))

Abstract

Stable isotope labeling with amino acids in cell culture (SILAC) is considered the most accurate method for proteome quantification by mass spectrometry. As it relies on active protein translation, it was traditionally limited to cells in culture and was not applicable to tissues. We have previously developed the super-SILAC mix, which is a mixture of several cell lines that serves as an internal spike-in standard for the study of human tumor tissue. The super-SILAC mix greatly improves the quantification accuracy while lowering error rates, and it is a simple, economic, and robust technique. Here we describe the design and application of super-SILAC to a broad range of biological systems, for basic biological research as well as clinical one.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Gstaiger M, Aebersold R (2009) Applying mass spectrometry-based proteomics to genetics, genomics and network biology. Nat Rev Genet 10:617–627

    Article  CAS  PubMed  Google Scholar 

  2. Cravatt BF, Simon GM, Yates JR III (2007) The biological impact of mass-spectrometry-based proteomics. Nature 450:991–1000

    Article  CAS  PubMed  Google Scholar 

  3. Mann M, Kelleher NL (2008) Precision proteomics: the case for high resolution and high mass accuracy. Proc Natl Acad Sci U S A 105:18132–18138

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Cox J, Mann M (2011) Quantitative, high-resolution proteomics for data-driven systems biology. Annu Rev Biochem 80:273–299

    Article  CAS  PubMed  Google Scholar 

  5. Choudhary C, Mann M (2010) Decoding signalling networks by mass spectrometry-based proteomics. Nat Rev Mol Cell Biol 11:427–439

    Article  CAS  PubMed  Google Scholar 

  6. Mallick P, Kuster B (2010) Proteomics: a pragmatic perspective. Nat Biotechnol 28:695–709

    Article  CAS  PubMed  Google Scholar 

  7. Ong SE, Mann M (2005) Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol 1:252–262

    Article  CAS  PubMed  Google Scholar 

  8. Ishihama Y, Sato T, Tabata T et al (2005) Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards. Nat Biotechnol 23:617–621

    Article  CAS  PubMed  Google Scholar 

  9. Geiger T, Cox J, Ostasiewicz P et al (2010) Super-SILAC mix for quantitative proteomics of human tumor tissue. Nat Methods 7:383–385

    Article  CAS  PubMed  Google Scholar 

  10. Deeb SJ, D’souza RC, Cox J et al (2012) Super-SILAC allows classification of diffuse large B-cell lymphoma subtypes by their protein expression profiles. Mol Cell Proteomics 11:77–89

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Geiger T, Wisniewski JR, Cox J et al (2011) Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics. Nat Protoc 6:147–157

    Article  CAS  PubMed  Google Scholar 

  12. Wisniewski JR, Zougman A, Nagaraj N et al (2009) Universal sample preparation method for proteome analysis. Nat Methods 6:359–362

    Article  CAS  PubMed  Google Scholar 

  13. Wisniewski JR, Zougman A, Mann M (2009) Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J Proteome Res 8:5674–5678

    Article  CAS  PubMed  Google Scholar 

  14. Cox J, Matic I, Hilger M et al (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc 4:698–705

    Article  CAS  PubMed  Google Scholar 

  15. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372

    Article  CAS  PubMed  Google Scholar 

  16. Britton HTS, Robinson RA (1931) CXCVIII.-Universal buffer solutions and the dissociation constant of veronal. J Chem Soc (Resumed): 1456–1462

    Google Scholar 

  17. Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906

    Article  CAS  PubMed  Google Scholar 

  18. Bendall SC, Hughes C, Stewart MH et al (2008) Prevention of amino acid conversion in SILAC experiments with embryonic stem cells. Mol Cell Proteomics 7:1587–1597

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Van Hoof D, Pinkse MW, Oostwaard DW et al (2007) An experimental correction for arginine-to-proline conversion artifacts in SILAC-based quantitative proteomics. Nat Methods 4:677–678

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Tamar Geiger .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Pozniak, Y., Geiger, T. (2014). Design and Application of Super-SILAC for Proteome Quantification. In: Warscheid, B. (eds) Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). Methods in Molecular Biology, vol 1188. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1142-4_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1142-4_20

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1141-7

  • Online ISBN: 978-1-4939-1142-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics