iTRAQ Reagent-Based Quantitative Proteomic Analysis on a Linear Ion Trap Mass Spectrometer

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.
Keywords: quantitative proteomics • iTRAQ • linear ion trap • pulsed-Q-dissociation