Modulation of Phosphopeptide Fragmentation via Dual Spray Ion/Ion Reactions Using a Sulfonate-Incorporating Reagent

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• 作者：Victoria C. Cotham ; William M. McGee ; Jennifer S. Brodbelt
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：August 16, 2016
• 年：2016
• 卷：88
• 期：16
• 页码：8158-8165
• 全文大小：457K
• 年卷期：0
• ISSN：1520-6882

文摘

The labile nature of phosphoryl groups has presented a long-standing challenge for the characterization of protein phosphorylation via conventional mass spectrometry-based bottom-up proteomics methods. Collision-induced dissociation (CID) causes preferential cleavage of the phospho-ester bond of peptides, particularly under conditions of low proton mobility, and results in the suppression of sequence-informative fragmentation that often prohibits phosphosite determination. In the present study, the fragmentation patterns of phosphopeptides are improved through ion/ion-mediated peptide derivatization with 4-formyl-1,3-benezenedisulfonic acid (FBDSA) anions using a dual spray reactor. This approach exploits the strong electrostatic interactions between the sulfonate moieties of FBDSA and basic sites to facilitate gas-phase bioconjugation and to reduce charge sequestration and increase the yield of phosphate-retaining sequence ions upon CID. Moreover, comparative CID fragmentation analysis between unmodified phosphopeptides and those modified online with FBDSA or in solution via carbamylation and 4-sulfophenyl isothiocyanate (SPITC) provided evidence for sulfonate interference with charge-directed mechanisms that result in preferential phosphate elimination. Our results indicate the prominence of charge-directed neighboring group participation reactions involved in phosphate neutral loss, and the implementation of ion/ion reactions in a dual spray reactor setup provides a means to disrupt the interactions by competing hydrogen-bonding interactions between sulfonate groups and the side chains of basic residues.