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HiRIEF LC-MS enables deep proteome coverage and unbiased proteogenomics

Nature Methods volume 11, pages 5962 (2014) | Download Citation

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.

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References

  1. 1.

    , & Mol. Biosyst. 7, 284–291 (2011).

  2. 2.

    et al. Mol. Cell. Proteomics 10, M111.011627 (2011).

  3. 3.

    et al. Genome Biol. 7, R35 (2006).

  4. 4.

    & Proteomics 11, 1086–1093 (2011).

  5. 5.

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

  6. 6.

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

  7. 7.

    et al. Nat. Methods 9, 1207–1211 (2012).

  8. 8.

    Mol. Syst. Biol. 3, 102 (2007).

  9. 9.

    , , , & PLoS ONE 5, e8949 (2010).

  10. 10.

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

  11. 11.

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

  12. 12.

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

  13. 13.

    , , & Mol. Cell. Proteomics 5, 1968–1974 (2006).

  14. 14.

    , & Proteomics 7, 1746–1752 (2007).

  15. 15.

    , , , & J. Biomol. Tech. 16, 181–189 (2005).

  16. 16.

    , & Cell 147, 789–802 (2011).

  17. 17.

    et al. Cell 149, 1622–1634 (2012).

  18. 18.

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

  19. 19.

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

  20. 20.

    et al. Nature 478, 476–482 (2011).

  21. 21.

    , & J. Proteome Res. 8, 5674–5678 (2009).

  22. 22.

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

  23. 23.

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

  24. 24.

    , , , & Electrophoresis 29, 2768–2778 (2008).

  25. 25.

    , , , & J. Proteome Res. 7, 3022–3027 (2008).

  26. 26.

    , , , & Nat. Methods 4, 923–925 (2007).

  27. 27.

    et al. Genome Biol. 13, 418 (2012).

  28. 28.

    et al. Genome Res. 12, 996–1006 (2002).

  29. 29.

    , , , & Mol. Cell. Proteomics 11, M111.014050 (2012).

  30. 30.

    et al. Genome Med. 4, 86 (2012).

  31. 31.

    , , & PLoS ONE 6, e21910 (2011).

  32. 32.

    , , & PLoS Comput. Biol. 5, e1000598 (2009).

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

    • Rui M M Branca
    • , Lukas M Orre
    •  & Henrik J Johansson

    These authors contributed equally to this work.

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

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Janne Lehtiö.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15

Excel files

  1. 1.

    Supplementary Table 1

    Conventional proteomics performance as measured by number of PSMs, peptides, protein groups and corresponding genes.

  2. 2.

    Supplementary Table 2

    Novel peptides identified by proteogenomics in H. sapiens and supporting evidence.

  3. 3.

    Supplementary Table 3

    Novel peptides identified by proteogenomics in M. musculus and supporting evidence.

  4. 4.

    Supplementary Table 4

    Proteins identified by conventional proteomics in H. sapiens.

  5. 5.

    Supplementary Table 5

    Proteins identified by conventional proteomics in M. musculus.

Zip files

  1. 1.

    Supplementary Data 1

    Annotations of MS2 spectra pertaining to the novel peptides.

  2. 2.

    Supplementary Data 2

    Custom tracks for data visualization in the UCSC genome browser.

  3. 3.

    Supplementary Software

    PredpI algorithm

About this article

Publication history

Received

Accepted

Published

DOI

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

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