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 set-up provides a means to disrupt the interactions by competing hydrogen-bonding interactions between sulfonate groups and the side-chains of basic residues.

Phosphopeptide Fragmentation and Site Localization by Mass Spectrometry: An Update

“…36 This is in accordance with the calculations made by Rožman, revealing that HPO 3 loss can compete with H 3 PO 4 loss under limited proton mobility conditions. 31 In an effort to limit the neutral loss extent, a chemical derivatization strategy mitigating charge-directed mechanisms leading to phosphate neutral loss was developed, 38 while addition of a dinuclear gallium complex improved the stability of the phospho-ester bond during CID. 39…”

The Role of Electron Transfer Dissociation in Modern Proteomics

“… 324 , 325 Ion–ion reactions were also harnessed by Brodbelt and co-workers to derivative peptides with 4-formyl-1,3-benezenedisulfonic acid (FBDSA) anions to improve collisional dissociation of phosphopeptides and UVPD fragmentation efficiencies. 326 , 327 …”

Gas‐phase rearrangement reaction of Schiff‐base‐modified peptide ions

“…Gas‐phase Schiff base formation has been demonstrated as an approach to provide structural information complementary to that derived from unmodified species in the tandem mass spectrometry of polypeptide ions . It involves the reactivity between a peptide primary amine group, either on the N‐terminus or the lysine side chain, and the formyl group on the reagent ion.…”

“…Fragmentation of the unmodified phosphopeptide often results in loss of the phosphate functionality and thereby precludes identification of the phosphate position. In the case of Schiff‐base‐modified peptides, on the other hand, the sulfonate group on the reagent disrupts the phosphate neutral loss process and stabilizes the phosphate group when collision‐induced dissociation (CID) is applied …”

“…Studies of Schiff‐base‐modified polypeptide ions via tandem mass spectrometry using CID have resulted predominantly in b/y‐type fragment ions, in analogy with unmodified ions . However, in this study, we focus on fragmentation products generated via CID that are unique to the Schiff‐base‐modified peptide.…”