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

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• 作者：Timothy J. Griffin ; Hongwei Xie ; Sricharan Bandhakavi ; Jonathan Popko ; Archana Mohan ; John V. Carlis ; LeeAnn Higgins
• 刊名：Journal of Proteome Research
• 出版年：2007
• 出版时间：November 2007
• 年：2007
• 卷：6
• 期：11
• 页码：4200 - 4209
• 全文大小：239K
• 年卷期：v.6,no.11(November 2007)
• ISSN：1535-3907

文摘

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