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Journal of the American Society for Mass Spectrometry

, Volume 19, Issue 9, pp 1255–1262 | Cite as

Peptide and protein quantification using iTRAQ with electron transfer dissociation

  • Doug Phanstiel
  • Yi Zhang
  • Jarrod A. Marto
  • Joshua J. CoonEmail author
Article

Abstract

Electron transfer dissociation (ETD) has become increasingly used in proteomic analyses due to its complementarity to collision-activated dissociation (CAD) and its ability to sequence peptides with post-translation modifications (PTMs). It was previously unknown, however, whether ETD would be compatible with a commonly employed quantification technique, isobaric tags for relative and absolute quantification (iTRAQ), since the fragmentation mechanisms and pathways of ETD differ significantly from CAD. We demonstrate here that ETD of iTRAQ labeled peptides produces c-and ż-type fragment ions as well as reporter ions that are unique from those produced by CAD. Exact molecular formulas of product ions were determined by ETD fragmentation of iTRAQ-labeled synthetic peptides followed by high mass accuracy orbitrap mass analysis. These experiments revealed that ETD cleavage of the N-Cα bond of the iTRAQ tag results in fragment ions that could be used for quantification. Synthetic peptide work demonstrates that these fragment ions provide up to three channels of quantification and that the quality is similar to that provided by beam-type CAD. Protein standards were used to evaluate peptide and protein quantification of iTRAQ labeling in conjunction with ETD, beam-type CAD, and pulsed Q dissociation (PQD) on a hybrid ion trap-orbitrap mass spectrometer. For reporter ion intensities above a certain threshold all three strategies provided reliable peptide quantification (average error < 10%). Approximately 36%, 8%, and 16% of scans identified fall below this threshold for ETD, HCD, and PQD, respectively. At the protein level, average errors were 2.3%, 1.7%, and 3.6% for ETD, HCD, and PQD, respectively.

Keywords

Electron Transfer Dissociation iTRAQ Label Orbitrap Mass Analysis Electron Transfer Dissociation Fragmentation Electron Transfer Dissociation Reaction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© American Society for Mass Spectrometry 2008

Authors and Affiliations

  • Doug Phanstiel
    • 1
  • Yi Zhang
    • 3
  • Jarrod A. Marto
    • 3
    • 4
  • Joshua J. Coon
    • 1
    • 2
    Email author
  1. 1.Department of ChemistryUniversity of WisconsinMadisonUSA
  2. 2.Department of Biomolecular ChemistryUniversity of WisconsinMadisonUSA
  3. 3.Department of Cancer Biology and Blais Proteomics CenterDana-Farber Cancer InstituteBostonUSA
  4. 4.Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBostonUSA

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