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:

HiRIEF LC-MS enables deep proteome coverage and unbiased proteogenomics

Nature Methods volume 11, pages 59–62 (2014)Cite this article

Subjects

Abstract

We present a liquid chromatography–mass spectrometry (LC-MS)-based method permitting unbiased (gene prediction–independent) genome-wide discovery of protein-coding loci in higher eukaryotes. Using high-resolution isoelectric focusing (HiRIEF) at the peptide level in the 3.7–5.0 pH range and accurate peptide isoelectric point (pI) prediction, we probed the six-reading-frame translation of the human and mouse genomes and identified 98 and 52 previously undiscovered protein-coding loci, respectively. The method also enabled deep proteome coverage, identifying 13,078 human and 10,637 mouse proteins.

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: HiRIEF LC-MS enables unbiased proteogenomics in higher eukaryotes.
Figure 2: Analysis of distribution of novel peptides into different categories.
Figure 3: New gene models.

Similar content being viewed by others

References

  1. Krug, K., Nahnsen, S. & Macek, B. Mol. Biosyst. 7, 284–291 (2011).

    Article  CAS  Google Scholar 

  2. Kelkar, D.S. et al. Mol. Cell. Proteomics 10, M111.011627 (2011).

    Article  Google Scholar 

  3. Fermin, D. et al. Genome Biol. 7, R35 (2006).

    Article  Google Scholar 

  4. Granholm, V. & Käll, L. Proteomics 11, 1086–1093 (2011).

    Article  CAS  Google Scholar 

  5. Baerenfaller, K. et al. Science 320, 938–941 (2008).

    Article  CAS  Google Scholar 

  6. Brosch, M. et al. Genome Res. 21, 756–767 (2011).

    Article  CAS  Google Scholar 

  7. Evans, V.C. et al. Nat. Methods 9, 1207–1211 (2012).

    Article  CAS  Google Scholar 

  8. Edwards, N.J. Mol. Syst. Biol. 3, 102 (2007).

    Article  Google Scholar 

  9. Bitton, D.A., Smith, D.L., Connolly, Y., Scutt, P.J. & Miller, C.J. PLoS ONE 5, e8949 (2010).

    Article  Google Scholar 

  10. Sevinsky, J.R. et al. J. Proteome Res. 7, 80–88 (2008).

    Article  CAS  Google Scholar 

  11. Tanner, S. et al. Genome Res. 17, 231–239 (2007).

    Article  CAS  Google Scholar 

  12. Beck, M. et al. Mol. Syst. Biol. 7, 549 (2011).

    Article  Google Scholar 

  13. Hörth, P., Miller, C.A., Preckel, T. & Wenz, C. Mol. Cell. Proteomics 5, 1968–1974 (2006).

    Article  Google Scholar 

  14. Lengqvist, J., Uhlen, K. & Lehtio, J. Proteomics 7, 1746–1752 (2007).

    Article  CAS  Google Scholar 

  15. Cargile, B.J., Sevinsky, J.R., Essader, A.S., Stephenson, J.L. Jr. & Bundy, J.L. J. Biomol. Tech. 16, 181–189 (2005).

    PubMed  PubMed Central  Google Scholar 

  16. Ingolia, N.T., Lareau, L.F. & Weissman, J.S. Cell 147, 789–802 (2011).

    Article  CAS  Google Scholar 

  17. Kalyana-Sundaram, S. et al. Cell 149, 1622–1634 (2012).

    Article  CAS  Google Scholar 

  18. Stedman, H.H. et al. Nature 428, 415–418 (2004).

    Article  CAS  Google Scholar 

  19. Djebali, S. et al. Nature 489, 101–108 (2012).

    Article  CAS  Google Scholar 

  20. Lindblad-Toh, K. et al. Nature 478, 476–482 (2011).

    Article  CAS  Google Scholar 

  21. Wiśniewski, J.R., Zougman, A. & Mann, M. J. Proteome Res. 8, 5674–5678 (2009).

    Article  Google Scholar 

  22. Reiter, L. et al. Mol. Cell. Proteomics 8, 2405–2417 (2009).

    Article  CAS  Google Scholar 

  23. Bjellqvist, B. et al. Electrophoresis 14, 1023–1031 (1993).

    Article  CAS  Google Scholar 

  24. Cargile, B.J., Sevinsky, J.R., Essader, A.S., Eu, J.P. & Stephenson, J.L. Electrophoresis 29, 2768–2778 (2008).

    Article  CAS  Google Scholar 

  25. Park, C.Y., Klammer, A.A., Käll, L., MacCoss, M.J. & Noble, W.S. J. Proteome Res. 7, 3022–3027 (2008).

    Article  CAS  Google Scholar 

  26. Käll, L., Canterbury, J.D., Weston, J., Noble, W.S. & MacCoss, M.J. Nat. Methods 4, 923–925 (2007).

    Article  Google Scholar 

  27. Stamatoyannopoulos, J.A. et al. Genome Biol. 13, 418 (2012).

    Article  Google Scholar 

  28. Kent, W.J. et al. Genome Res. 12, 996–1006 (2002).

    Article  CAS  Google Scholar 

  29. Geiger, T., Wehner, A., Schaab, C., Cox, J. & Mann, M. Mol. Cell. Proteomics 11, M111.014050 (2012).

    Article  Google Scholar 

  30. Akan, P. et al. Genome Med. 4, 86 (2012).

    Article  CAS  Google Scholar 

  31. Stranneheim, H., Werne, B., Sherwood, E. & Lundeberg, J. PLoS ONE 6, e21910 (2011).

    Article  CAS  Google Scholar 

  32. Ramsköld, D., Wang, E.T., Burge, C.B. & Sandberg, R. PLoS Comput. Biol. 5, e1000598 (2009).

    Article  Google Scholar 

Download references

Acknowledgements

Funding from the Swedish Research Council, Swedish Cancer Society, Stockholm's county council, Stockholm's cancer society and EU FP7 project GlycoHit is gratefully acknowledged. Support by BILS (Bioinformatics Infrastructure for Life Sciences) and J. Boekel in publishing the MS raw files is gratefully acknowledged. We thank the SciLifeLab genomics facility for experimental support and J. Lundeberg for the A431 sequence data. We thank E. Bereczki (Karolinska Institutet) for the kind gift of the N2A cell line. We thank K. Lindblad-Toh for critical reading of the manuscript. We acknowledge the late B. Bjellqvist for his early contribution in the development of IPG-IEF and peptide pI prediction.

Author information

Author notes

  1. Rui M M Branca, Lukas M Orre and Henrik J Johansson: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Oncology-Pathology, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden

    Rui M M Branca, Lukas M Orre, Henrik J Johansson, Åsa Pérez-Bercoff, Jenny Forshed & Janne Lehtiö

  2. Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Stockholm, Sweden

    Viktor Granholm

  3. Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Science for Life Laboratory, Stockholm University, Stockholm, Sweden

    Mikael Huss

  4. School of Biotechnology, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden

    Lukas Käll

  5. Swedish e-Science Resource Center, KTH Royal Institute of Technology, Stockholm, Sweden

    Lukas Käll

Authors
  1. Rui M M Branca
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lukas M Orre
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Henrik J Johansson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Viktor Granholm
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mikael Huss
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Åsa Pérez-Bercoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jenny Forshed
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lukas Käll
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Janne Lehtiö
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.L., R.M.M.B., L.M.O., L.K. and H.J.J. conceived of and designed the experiments. R.M.M.B. and H.J.J. performed the IEF separations and MS analysis. M.H., L.M.O. and R.M.M.B. performed the data analysis of RNA-seq experiments. R.M.M.B. performed the peptide pI calculations. L.K., J.L., R.M.M.B. and V.G. designed the database restriction workflow and performed the 6FT searches. L.K. and V.G. designed the novel-only TDA approach. Å.P.-B. performed the single-nucleotide polymorphism data analysis and calculated Ensembl annotation statistics. R.M.M.B., L.M.O., H.J.J. and J.F. performed proteomics data analysis. R.M.M.B., L.M.O. and J.L. wrote the manuscript. All authors were involved in discussion of the manuscript and approved the final manuscript.

Corresponding author

Correspondence to Janne Lehtiö.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15 (PDF 1952 kb)

Supplementary Table 1

Conventional proteomics performance as measured by number of PSMs, peptides, protein groups and corresponding genes. (XLSX 10 kb)

Supplementary Table 2

Novel peptides identified by proteogenomics in H. sapiens and supporting evidence. (XLSX 103 kb)

Supplementary Table 3

Novel peptides identified by proteogenomics in M. musculus and supporting evidence. (XLSX 35 kb)

Supplementary Table 4

Proteins identified by conventional proteomics in H. sapiens. (XLSX 4938 kb)

Supplementary Table 5

Proteins identified by conventional proteomics in M. musculus. (XLSX 895 kb)

Supplementary Data 1

Annotations of MS2 spectra pertaining to the novel peptides. (ZIP 6962 kb)

Supplementary Data 2

Custom tracks for data visualization in the UCSC genome browser. (ZIP 766 kb)

Supplementary Software

PredpI algorithm (ZIP 2988 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Cite this article

Branca, R., Orre, L., Johansson, H. et al. HiRIEF LC-MS enables deep proteome coverage and unbiased proteogenomics. Nat Methods 11, 59–62 (2014). https://doi.org/10.1038/nmeth.2732

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2732

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