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.

Figure 1

CID product ion mass spectra of RRLIEDAEpYAARG-NH₂ (2+) before and after Schiff base modification: (a) MS CID mass spectrum of unlabeled peptide and (b) MS CID mass spectrum following online dual spray reactor-initiated derivatization. The addition of “◆” to the label indicates covalent FBDSA Schiff base modification and “-P” indicates loss of phosphate.

Figure 2

CID product ion mass spectra of GGGPApTPKKAKKL (2+) before and after Schiff base modification: (a) MS CID mass spectrum of unlabeled peptide and (b) MS CID mass spectrum following online dual spray reactor-initiated derivatization. The addition of “◆” to the label indicates covalent FBDSA Schiff base modification and “-P” indicates loss of phosphate. Ions shown in blue arise from lysine modification, while those shown in black may arise from either lysine or N-terminally labeled species.

Figure 3

Percent of total product ion abundance arising from neutral loss of phosphate from the precursor ion of unmodified and Schiff based-labeled phosphopeptides subjected to CID.

Figure 4

Variable energy CID analysis of unmodified and N-terminally carbamylated, SPITC-and FBDSA-modified (a) RRLIEDAEpYAARG-NH2 (2+) and (b) RQpSVELHSPQSLPR (2+). Normalized precursor abundances are plotted as a function of increasing collision energy. The CID product ion spectra are shown for RQpSVELHSPQSLPR (2+) in the following states: (c) unmodified, (d) carbamylated (*), and (e) FBDSA Schiff base modified (◆). The addition of “-P” to the label indicates loss of phosphate.

Scheme 1

(a) Proposed mechanism for charge-directed neutral loss of phosphate for an unmodified phosphopeptide. Proposed competing dissociation pathways for FBDSA-labeled phosphopeptides with (b) and without (c) sulfonate-modulated suppression of charge-directed neutral loss of phosphate.