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A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS)

Nature Protocols volume 4, pages 870–877 (2009)Cite this article

Abstract

The application of a targeted mass spectrometric workflow to the sensitive identification of post-translational modifications is described. This protocol employs multiple reaction monitoring (MRM) to search for all putative peptides specifically modified in a target protein. Positive MRMs trigger an MS/MS experiment to confirm the nature and site of the modification. This approach, termed MIDAS (MRM-initiated detection and sequencing), is more sensitive than approaches using neutral loss scanning or precursor ion scanning methodologies, due to a more efficient use of duty cycle along with a decreased background signal associated with MRM. We describe the use of MIDAS for the identification of phosphorylation, with a typical experiment taking just a couple of hours from obtaining a peptide sample. With minor modifications, the MIDAS method can be applied to other protein modifications or unmodified peptides can be used as a MIDAS target.

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Figure 1: The MIDAS workflow.
Figure 2: Results of a typical MIDAS analysis.

References

  1. Walsh, C.T. Posttranslational Modifications of Proteins: Expanding Nature's Inventory (Roberts and Co., Greenwood, CO, 2006).

    Google Scholar 

  2. Andon, N.L., Eckert, D., Yates, J.R. III. & Haynes, P.A. High-throughput functional affinity purification of mannose binding proteins from Oryza sativa . Proteomics 3, 1270–1278 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Iwabata, H., Yoshida, M. & Komatsu, Y. Proteomic analysis of organ-specific post-translational lysine-acetylation and -methylation in mice by use of anti-acetyllysine and -methyllysine mouse monoclonal antibodies. Proteomics 5, 4653–4664 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Posewitz, M.C. & Tempst, P. Immobilized gallium (III) affinity chromatography of phosphopeptides. Anal. Chem. 71, 2883–2892 (1999).

    Article  CAS  PubMed  Google Scholar 

  5. Montagnoli, A. et al. Identification of Mcm2 phosphorylation sites by S-phase-regulating kinases. J. Biol. Chem. 281, 10281–10290 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Schlosser, A., Pipkorn, R., Bossemeyer, D. & Lehmann, W.D. Analysis of protein phosphorylation by a combination of elastase digestion and neutral loss tandem mass spectrometry. Anal. Chem. 73, 170–176 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Steen, H., Kuster, B., Fernandez, M., Pandey, A. & Mann, M. Detection of tyrosine phosphorylated peptides by precursor ion scanning quadrupole TOF mass spectrometry in positive ion mode. Anal. Chem. 73, 1440–1448 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Unwin, R.D. et al. Multiple reaction monitoring to identify sites of protein phosphorylation with high sensitivity. Mol. Cell Proteomics 4, 1134–1144 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Griffiths, J.R. et al. The application of a hypothesis-driven strategy to the sensitive detection and location of acetylated lysine residues. J. Am. Soc. Mass Spectrom. 18, 1423–1428 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Mollah, S. et al. Targeted mass spectrometric strategy for global mapping of ubiquitination on proteins. Rapid Commun. Mass Spectrom. 21, 3357–3364 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Ciccimaro, E., Hevko, J. & Blair, I.A. Analysis of phosphorylation sites on focal adhesion kinase using nanospray liquid chromatography/multiple reaction monitoring mass spectrometry. Rapid Commun. Mass Spectrom 20, 3681–3692 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Welman, A., Griffiths, J.R., Whetton, A.D. & Dive, C. Protein kinase C delta is phosphorylated on five novel Ser/Thr sites following inducible overexpression in human colorectal cancer cells. Protein Sci. 16, 2711–2715 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Pierce, A. et al. THOC5 spliceosome protein: a target for leukaemogenic tyrosine kinases that affects inositol lipid turnover. Br. J. Haematol. 141, 641–650 (2008).

    Article  CAS  PubMed  Google Scholar 

  14. Traweger, A. et al. Protein phosphatase 1 regulates the phosphorylation state of the polarity scaffold Par-3. Proc. Natl Acad. Sci. USA 105, 10402–10407 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. McCoy, C.E. et al. Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS. Biochem. J. 402, 491–501 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Anderson, L. & Hunter, C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol. Cell Proteomics 5, 573–588 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Unwin, R.D. et al. Global effects of BCR/ABL and TEL/PDGFRâ expression on the proteome and phosphoproteome: identification of the Rho pathway as a target of BCR/ABL. J. Biol. Chem. 280, 6316–6326 (2005).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This study was supported by Leukaemia Research UK and BBSRC.

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  1. Stem Cell and Leukaemia Proteomics Laboratory, Faculty of Medicine and Human Sciences, University of Manchester, Manchester, UK

    Richard D Unwin, John R Griffiths & Anthony D Whetton

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  1. Richard D Unwin
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Correspondence to Richard D Unwin.

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Unwin, R., Griffiths, J. & Whetton, A. A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS). Nat Protoc 4, 870–877 (2009). https://doi.org/10.1038/nprot.2009.57

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