iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer

Timothy J. Griffin, Hongwei Xie, Sricharan Bandhakavi, Jonathan Popko, Archana Mohan, John V. Carlis, Lee Ann Higgins

Research output: Contribution to journalArticlepeer-review

121 Scopus citations

Abstract

For proteomic analysis using tandem mass spectrometry, linear ion trap instruments provide unsurpassed sensitivity but unreliably detect low mass peptide fragments, precluding their use with iTRAQ reagent-labeled samples. Although the popular LTQ linear ion trap supports analyzing iTRAQ reagent-labeled peptides via pulsed Q dissociation, PQD, its effectiveness remains questionable. Using a standard mixture, we found careful tuning of relative collision energy necessary for fragmenting iTRAQ reagent-labeled peptides, and increasing microscan acquisition and repeat count improves quantification but identifies somewhat fewer peptides. We developed software to calculate abundance ratios via summing reporter ion intensities across spectra matching to each protein, thereby providing maximized accuracy. Testing found that results closely corresponded between analysis using optimized LTQ-PQD settings plus our software and using a Qstar instrument. Thus, we demonstrate the effectiveness of LTQ-PQD analyzing iTRAQ reagent-labeled peptides, and provide guidelines for successful quantitative proteomic studies.

Original languageEnglish (US)
Pages (from-to)4200-4209
Number of pages10
JournalJournal of Proteome Research
Volume6
Issue number11
DOIs
StatePublished - Nov 2007

Keywords

  • Linear ion trap
  • Pulsed-Q-dissociation
  • Quantitative proteomics
  • iTRAQ

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