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Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics

Nature Protocols volume 6, pages 147–157 (2011)Cite this article

Abstract

Mass spectrometry (MS)-based proteomics is increasingly applied in a quantitative format, often based on labeling of samples with stable isotopes that are introduced chemically or metabolically. In the stable isotope labeling by amino acids in cell culture (SILAC) method, two cell populations are cultured in the presence of heavy or light amino acids (typically lysine and/or arginine), one of them is subjected to a perturbation, and then both are combined and processed together. In this study, we describe a different approach—the use of SILAC as an internal or 'spike-in' standard—wherein SILAC is only used to produce heavy labeled reference proteins or proteomes. These are added to the proteomes under investigation after cell lysis and before protein digestion. The actual experiment is therefore completely decoupled from the labeling procedure. Spike-in SILAC is very economical, robust and in principle applicable to all cell- or tissue-based proteomic analyses. Applications range from absolute quantification of single proteins to the quantification of whole proteomes. Spike-in SILAC is especially advantageous when analyzing the proteomes of whole tissues or organisms. The protocol describes the quantitative analysis of a tissue sample relative to super-SILAC spike-in, a mixture of five SILAC-labeled cell lines that accurately represents the tissue. It includes the selection and preparation of the spike-in SILAC standard, the sample preparation procedure, and analysis and evaluation of the results.

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Figure 1: The workflow of classical SILAC experiment versus spike-in SILAC standard.
Figure 2: Applications of spike-in SILAC standards.
Figure 3: Evaluation of the quality of the spike-in standard.

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Acknowledgements

We thank P. Roerth for suggesting the term 'spike-in SILAC'. T.G. is supported by the Humboldt Foundation. This project was supported by the European Commission's 7th Framework Program PROteomics SPECification in Time and Space (PROSPECTS, HEALTH-F4-2008-021,648).

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Authors and Affiliations

  1. Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany

    Tamar Geiger, Jacek R Wisniewski, Juergen Cox, Sara Zanivan & Matthias Mann

  2. Department of Cardiac Development and Remodeling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

    Marcus Kruger

  3. Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan

    Yasushi Ishihama

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  1. Tamar Geiger
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Contributions

T.G. developed the super-SILAC mix protocol. J.R.W. contributed the FASP and SAX protocols. J.C. developed MaxQuant and adapted it to spike-in SILAC. S.Z. contributed to the development, analysis and maintenance of the SILAC mice. M.K. developed and analyzed the SILAC mice. Y.I. contributed to the use of SILAC in a cell line as a spike-in standard. M.M. initiated and supervised all of these projects. T.G. and M.M. wrote the manuscript. All authors revised the manuscript.

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Correspondence to Matthias Mann.

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Geiger, T., Wisniewski, J., Cox, J. et al. Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics. Nat Protoc 6, 147–157 (2011). https://doi.org/10.1038/nprot.2010.192

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