title: Modulation of Phosphopeptide Fragmentation via Dual Spray Ion/Ion Reactions Using a Sulfonate-Incorporating Reagent.
abstract: 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.
authors.affiliation: Department of Chemistry, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Victoria C
authors.initials: VC
authors.lastname: Cotham
authors.affiliation: Department of Chemistry, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: William M
authors.initials: WM
authors.lastname: McGee
authors.affiliation: Department of Chemistry, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Brodbelt
mesh_terms: D000595:Amino Acid Sequence; D000838:Anions / Q000737:chemistry; D001557:Benzenesulfonates / Q000737:chemistry*; D017879:Isothiocyanates / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry; D012545:Schiff Bases / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D055672:Static Electricity
chemical_list: D000838:Anions; D001557:Benzenesulfonates; D017879:Isothiocyanates; D010748:Phosphopeptides; D012545:Schiff Bases; C015308:4-isothiocyanatobenzenesulfonate; C098804:benzene 1,3-disulfonate