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Research Article

Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry

Henrik Molina, David M. Horn, Ning Tang, Suresh Mathivanan, and Akhilesh Pandey
  1. *McKusick-Nathans Institute for Genetic Medicine and Departments of Biological Chemistry, Pathology, and Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;
  2. †Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense 5230, Denmark;
  3. ‡Agilent Technologies, Santa Clara, CA 95052; and
  4. §Institute of Bioinformatics, International Tech Park, Bangalore 560 066, India

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PNAS February 13, 2007 104 (7) 2199-2204; https://doi.org/10.1073/pnas.0611217104
Henrik Molina
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David M. Horn
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Ning Tang
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Suresh Mathivanan
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Akhilesh Pandey
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  • For correspondence: pandey@jhmi.edu
  1. Communicated by Paul Talalay, Johns Hopkins University School of Medicine, Baltimore, MD, December 25, 2006 (received for review November 1, 2006)

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Abstract

Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique that provides a more comprehensive coverage of peptide sequences and posttranslational modifications. Here, we evaluated the use of ETD for a global phosphoproteome analysis. In all, we identified a total of 1,435 phosphorylation sites from human embryonic kidney 293T cells, of which 1,141 (≈80%) were not previously described. A detailed comparison of ETD and collision-induced dissociation (CID) modes showed that ETD identified 60% more phosphopeptides than CID, with an average of 40% more fragment ions that facilitated localization of phosphorylation sites. Although our data indicate that ETD is superior to CID for phosphorylation analysis, the two methods can be effectively combined in alternating ETD and CID modes for a more comprehensive analysis. Combining ETD and CID, from this single study, we were able to identify 80% of the known phosphorylation sites in >1,000 phosphorylated peptides analyzed. A hierarchical clustering of the identified phosphorylation sites allowed us to discover 15 phosphorylation motifs that have not been reported previously. Overall, ETD is an excellent method for localization of phosphorylation sites and should be an integral component of any strategy for comprehensive phosphorylation analysis.

  • bioinformatics
  • motifs
  • phosphorylation
  • signal transduction
  • systems biology

Footnotes

  • ¶To whom correspondence should be addressed. E-mail: pandey{at}jhmi.edu
  • Author contributions: H.M. and A.P. designed research; H.M., N.T., and S.M. performed research; H.M., D.M.H., S.M., and A.P. analyzed data; and H.M. and A.P. wrote the paper.

  • Conflict of interest statement: D.M.H. and N.T. are employees of Agilent Technologies. All other authors have declared no conflict of interest.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0611217104/DC1.

  • Abbreviations:
    CID,
    collision-induced dissociation;
    ETD,
    electron transfer dissociation;
    MS/MS,
    tandem MS;
    LC,
    liquid chromatography.
  • Freely available online through the PNAS open access option.

  • © 2007 by The National Academy of Sciences of the USA
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Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry
Henrik Molina, David M. Horn, Ning Tang, Suresh Mathivanan, Akhilesh Pandey
Proceedings of the National Academy of Sciences Feb 2007, 104 (7) 2199-2204; DOI: 10.1073/pnas.0611217104

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Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry
Henrik Molina, David M. Horn, Ning Tang, Suresh Mathivanan, Akhilesh Pandey
Proceedings of the National Academy of Sciences Feb 2007, 104 (7) 2199-2204; DOI: 10.1073/pnas.0611217104
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