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.

  • Brief Communication
  • Published:

Integrated proteomic analysis of post-translational modifications by serial enrichment

Nature Methods volume 10, pages 634–637 (2013)Cite this article

Subjects

Abstract

We report a mass spectrometry–based method for the integrated analysis of protein expression, phosphorylation, ubiquitination and acetylation by serial enrichments of different post-translational modifications (SEPTM) from the same biological sample. This technology enabled quantitative analysis of nearly 8,000 proteins and more than 20,000 phosphorylation, 15,000 ubiquitination and 3,000 acetylation sites per experiment, generating a holistic view of cellular signal transduction pathways as exemplified by analysis of bortezomib-treated human leukemia cells.

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: SEPTM enables deep, quantitative analysis of the PTM-ome of a biological sample.
Figure 2: Comparison of nonserial to serial enrichments for K(GG) and K(Ac) enrichments.

Similar content being viewed by others

References

  1. Olsen, J.V. et al. Sci. Signal. 3, ra3 (2010).

    Article  Google Scholar 

  2. Kim, W. et al. Mol. Cell 44, 325–340 (2011).

    Article  CAS  Google Scholar 

  3. Udeshi, N.D. et al. Mol. Cell Proteomics 12, 825–831 (2013).

    Article  CAS  Google Scholar 

  4. Choudhary, C. et al. Science 325, 834–840 (2009).

    Article  CAS  Google Scholar 

  5. Ficarro, S.B. et al. Anal. Chem. 81, 4566–4575 (2009).

    Article  CAS  Google Scholar 

  6. Pinkse, M.W., Lemeer, S. & Heck, A.J. Methods Mol. Biol. 753, 215–228 (2011).

    Article  CAS  Google Scholar 

  7. Kim, S.C. et al. Mol. Cell 23, 607–618 (2006).

    Article  CAS  Google Scholar 

  8. Xu, G., Paige, J.S. & Jaffrey, S.R. Nat. Biotechnol. 28, 868–873 (2010).

    Article  CAS  Google Scholar 

  9. Ong, S.E. et al. Mol. Cell Proteomics 1, 376–386 (2002).

    Article  CAS  Google Scholar 

  10. Mertins, P. et al. Mol. Cell Proteomics 11, M111.014423 (2012).

    Article  Google Scholar 

  11. Villén, J. & Gygi, S.P. Nat. Protoc. 3, 1630–1638 (2008).

    Article  Google Scholar 

  12. McNulty, D.E. & Annan, R.S. Mol. Cell Proteomics 7, 971–980 (2008).

    Article  CAS  Google Scholar 

  13. Wang, Y. et al. Proteomics 11, 2019–2026 (2011).

    Article  CAS  Google Scholar 

  14. Udeshi, N.D. et al. Mol. Cell Proteomics 11, 148–159 (2012).

    Article  CAS  Google Scholar 

  15. Schwartz, D. & Gygi, S.P. Nat. Biotechnol. 23, 1391–1398 (2005).

    Article  CAS  Google Scholar 

  16. Hunter, T. Mol. Cell 28, 730–738 (2007).

    Article  CAS  Google Scholar 

  17. Cheng, S.W. et al. Mol. Cell Biol. 32, 4691–4704 (2012).

    Article  CAS  Google Scholar 

  18. Yang, F., Shen, Y., Camp, D.G. II. & Smith, R.D. Expert Rev. Proteomics 9, 129–134 (2012).

    Article  CAS  Google Scholar 

  19. Rappsilber, J., Mann, M. & Ishihama, Y. Nat. Protoc. 2, 1896–1906 (2007).

    Article  CAS  Google Scholar 

  20. Cox, J. et al. J. Proteome Res. 10, 1794–1805 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the Broad Institute of MIT and Harvard and by the following grants from the US National Institutes of Health: grant U24CA160034 from the National Cancer Institute Clinical Proteomics Tumor Analysis Consortium Initiative (to S.A.C.) and grants HHSN268201000033C and R01HL096738 from the National Heart, Lung, and Blood Institute (to S.A.C.).

Author information

Authors and Affiliations

  1. The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Philipp Mertins, Jana W Qiao, Jinal Patel, Namrata D Udeshi, Karl R Clauser, D R Mani, Michael W Burgess, Michael A Gillette, Jacob D Jaffe & Steven A Carr

Authors
  1. Philipp Mertins
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jana W Qiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jinal Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Namrata D Udeshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Karl R Clauser
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. D R Mani
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Michael W Burgess
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michael A Gillette
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jacob D Jaffe
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Steven A Carr
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.M. and S.A.C. developed the SEPTM strategy and conceived of the study; P.M., J.W.Q. and J.P. performed experiments; P.M., J.W.Q., J.P., N.D.U., K.R.C., D.R.M., M.W.B., M.A.G., J.D.J. and S.A.C. contributed to experimental design and data analysis; and P.M. and S.A.C. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Philipp Mertins or Steven A Carr.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Combined PDF

Supplementary Figures 1–4 and Supplementary Tables 5–7 (PDF 747 kb)

Supplementary Table 1

List of all quantified proteins, pSTY-, K(GG)- and K(Ac)-sites that were quantified in at least two replicates in the high, medium and low coverage experiments. (XLSX 22886 kb)

Supplementary Table 2

List of all quantified peptides observed in the proteome analysis at high, medium and low coverage of one representative replicate (replicate 1). (XLSX 28756 kb)

Supplementary Table 3

List of all quantified peptides observed in the phosphoproteome, ubiquitinome and acetylome analyses at high, medium and low coverage of one representative replicate (replicate 1). (XLSX 22298 kb)

Supplementary Table 4

Ubiquitin- and acetyl-site tables for label-free and SILAC-based comparison of non-serial and serial enrichments. (XLSX 2662 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mertins, P., Qiao, J., Patel, J. et al. Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Methods 10, 634–637 (2013). https://doi.org/10.1038/nmeth.2518

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2518

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research