Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Absolute quantification of protein and post-translational modification abundance with stable isotope–labeled synthetic peptides

Nature Protocols volume 6, pages 175–186 (2011)Cite this article

Subjects

Abstract

In the analysis of biological systems, it is of interest to identify the components of the system and to monitor their changes in abundance under different conditions. The AQUA (for 'absolute quantification') method allows sensitive and specific targeted quantification of protein and post-translational modifications in complex protein mixtures using stable isotope–labeled peptides as internal standards. Each AQUA experiment is composed of two stages: method development and application to a biological scenario. In the method development stage, peptides from the protein of interest are chosen and then synthesized with stable isotopes such as 13C, 2H or 15N. The abundance of these internal standards and their endogenous counterparts can be measured by mass spectrometry with selected reaction monitoring or selected ion monitoring methods. Once an AQUA method is established, it can be rapidly applied to a wide range of biological samples, from tissue culture cells to human plasma and tissue. After AQUA peptide synthesis, the development, optimization and application of AQUA analyses to a specific biological problem can be achieved in ∼1 week. Here we demonstrate the usefulness of this method by monitoring both Polo-like kinase 1 (Plk1) protein abundance in multiple lung cancer cell lines and the extent of Plk1 activation loop phosphorylation (pThr-210) during release from S phase.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Scheme of AQUA workflow.
Figure 2: MS/MS spectra of the AQUA peptides for measuring Plk1 abundance and Plk1 T210 T-loop phosphorylation.
Figure 3: Comparison of LC-SRM and LC-SIM traces for 10 fmol of each AQUA peptide as well as their endogenous counterparts from a Plk1 immunoprecipitation.
Figure 4: Comparison of the abundance measurements by LC-SRM and LC-SIM for the AQUA peptides.
Figure 5: Plk1 expression in lung cancer cells lines.
Figure 6: Plk1 threonine T210 phosphorylation during release from S phase.

Similar content being viewed by others

References

  1. Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell Proteomics 1, 376–86 (2002).

    Article  CAS  Google Scholar 

  2. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–9 (1999).

    Article  CAS  Google Scholar 

  3. Ross, P.L. et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154–1169 (2004).

    Article  CAS  Google Scholar 

  4. Gerber, S.A., Rush, J., Stemman, O., Kirschner, M.W. & Gygi, S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. USA 100, 6940–6945 (2003).

    Article  CAS  Google Scholar 

  5. Beynon, R.J., Doherty, M.K., Pratt, J.M. & Gaskell, S.J. Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides. Nat. Methods 2, 587–589 (2005).

    Article  CAS  Google Scholar 

  6. Pratt, J.M. et al. Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes. Nat. Protoc. 1, 1029–1043 (2006).

    Article  CAS  Google Scholar 

  7. Langenfeld, E., Zanger, U.M., Jung, K., Meyer, H.E. & Marcus, K. Mass spectrometry-based absolute quantification of microsomal cytochrome P450 2D6 in human liver. Proteomics 9, 2313–2323 (2009).

    Article  CAS  Google Scholar 

  8. Mayya, V., Rezual, K., Wu, L., Fong, M.B. & Han, D.K. Absolute quantification of multisite phosphorylation by selective reaction monitoring mass spectrometry: determination of inhibitory phosphorylation status of cyclin-dependent kinases. Mol. Cell. Proteomics 5, 1146–1157 (2006).

    Article  CAS  Google Scholar 

  9. Kirkpatrick, D.S. et al. Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nat. Cell Biol. 8, 700–710 (2006).

    Article  CAS  Google Scholar 

  10. Xu, P. et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133–145 (2009).

    Article  CAS  Google Scholar 

  11. Deutsch, E.W., Lam, H. & Aebersold, R. PeptideAtlas: a resource for target selection for emerging targeted proteomics workflows. EMBO Rep. 9, 429–434 (2008).

    Article  CAS  Google Scholar 

  12. Mathivanan, S. et al. Human Proteinpedia enables sharing of human protein data. Nat. Biotechnol. 26, 164–167 (2008).

    Article  CAS  Google Scholar 

  13. Jones, P. et al. PRIDE: a public repository of protein and peptide identifications for the proteomics community. Nucleic Acids Res. 34, D659–D663 (2006).

    Article  CAS  Google Scholar 

  14. Mallick, P. et al. Computational prediction of proteotypic peptides for quantitative proteomics. Nat. Biotechnol. 25, 125–131 (2007).

    Article  CAS  Google Scholar 

  15. Picotti, P. et al. High-throughput generation of selected reaction-monitoring assays for proteins and proteomes. Nat. Methods 7, 43–46 (2010).

    Article  CAS  Google Scholar 

  16. MacLean, B. et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26, 966–968 (2010).

    Article  CAS  Google Scholar 

  17. Papaioannou, M.D. et al. Loss of Dicer in Sertoli cells has a major impact on the testicular proteome of mice. Mol. Cell Proteomics Published online; doi:10.1074/mcp.M900587-MCP200 (13 May 2010).

  18. Stemmann, O., Zou, H., Gerber, S.A., Gygi, S.P. & Kirschner, M.W. Dual inhibition of sister chromatid separation at metaphase. Cell 107, 715–726 (2001).

    Article  CAS  Google Scholar 

  19. Wolf, G. et al. Prognostic significance of polo-like kinase (PLK) expression in non-small cell lung cancer. Oncogene 14, 543–549 (1997).

    Article  CAS  Google Scholar 

  20. Seki, A., Coppinger, J.A., Jang, C.Y., Yates, J.R. & Fang, G. Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry. Science 320, 1655–1658 (2008).

    Article  CAS  Google Scholar 

  21. Macurek, L. et al. Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery. Nature 455, 119–123 (2008).

    Article  CAS  Google Scholar 

  22. Olsen, J.V. et al. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci. Signal. 3, ra3 (2010).

    Article  Google Scholar 

  23. Beausoleil, S.A. et al. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. USA 101, 12130–12135 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Cullati and D. Schweppe for helpful discussion and comments on the manuscript. This work was supported by the National Institutes of Health Grant P20-RR018787 from the IDeA Program of the National Center for Research Resources and the American Cancer Society Grant IRG-82-003-24 (to S.A.G.).

Author information

Authors and Affiliations

  1. Department of Genetics, Dartmouth Medical School and Norris Cotton Cancer Center, Lebanon, New Hampshire, USA

    Arminja N Kettenbach & Scott A Gerber

  2. Cell Signaling Technology, Danvers, Massachusetts, USA

    John Rush

Authors
  1. Arminja N Kettenbach
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John Rush
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Scott A Gerber
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.N.K. designed and conducted the experiments for Plk1 AQUA analysis, interpreted the data and wrote the paper; J.R. synthesized the Plk1 AQUA peptides used in this study; and S.A.G. conceived of the experiments for Plk1 AQUA analysis, interpreted the data and wrote the paper.

Corresponding author

Correspondence to Scott A Gerber.

Ethics declarations

Competing interests

Scott Gerber is a patent holder for AQUA technology, and receives royalty income from this patent. John Rush is an employee of Cell Signaling Technology, which has licensed the AQUA technology and sells AQUA peptides.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kettenbach, A., Rush, J. & Gerber, S. Absolute quantification of protein and post-translational modification abundance with stable isotope–labeled synthetic peptides. Nat Protoc 6, 175–186 (2011). https://doi.org/10.1038/nprot.2010.196

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.196

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing