Quantification of Competing H₃PO₄ Versus HPO₃ + H₂O Neutral Losses from Regioselective ¹⁸O-Labeled Phosphopeptides

Abstract

Abundant neutral losses of 98 Da are often observed upon ion trap CID-MS/MS of protonated phosphopeptide ions. Two competing fragmentation pathways are involved in this process, namely, the direct loss of H3PO4 from the phosphorylated residue and the combined losses of HPO3 and H2O from the phosphorylation site and from an additional site within the peptide, respectively. These competing pathways produce product ions with different structures but the same m/z values, potentially limiting the utility of CID-MS(3) for phosphorylation site localization. To quantify the relative contributions of these pathways and to determine the conditions under which each pathway predominates, we have examined the ion trap CID-MS/MS fragmentation of a series of regioselective (18)O-phosphate ester labeled phosphopeptides prepared using novel solution-phase amino acid synthesis and solid-phase peptide synthesis methodologies. By comparing the intensity of the -100 Da (-H3PO3 (18)O) versus -98 Da (-[HPO3 + H2O]) neutral loss product ions formed upon MS/MS, quantification of the two pathways was achieved. Factors that affect the extent of formation of the competing neutral losses were investigated, with the combined loss pathway predominantly occurring under conditions of limited proton mobility, and with increased combined losses observed for phosphothreonine compared with phosphoserine-containing peptides. The combined loss pathway was found to be less dominant under ion activation conditions associated with HCD-MS/MS. Finally, the contribution of carboxylic acid functional groups and backbone amide bonds to the water loss in the combined loss fragmentation pathway was determined via methyl esterification and by examination of a phosphopeptide lacking side-chain hydroxyl groups.

Large-Scale Examination of Factors Influencing Phosphopeptide Neutral Loss during Collision Induced Dissociation

“…Indeed, single replacements tend to slightly lower the observed neutral loss rather than increase it. This is in agreement with the observations of Cui, et al who found that mutating acidic residues did not significantly affect neutral loss [17]. It should be noted the presence of one acidic residue greatly increases the chances of finding more acidic residues due to the presence of acidophilic kinase motifs in biological data sets.…”

“…This has been proposed as the mechanism for neutral loss of HPO 3 in pTyr [36, 37]. Reactions of this kind would be compatible with previous experiments which indicate that a competing neutral loss reaction pathway exists in which HPO 3 is lost from the phosphoresidue concomitantly with the loss of water from elsewhere in the peptide [17]. This reaction would be isobaric and indistinguishable from neutral loss of phosphoric acid.…”

“…Competition for a charge-stabilizing partner could explain the strong negative correlations that are observed between the neutral loss pathways. The inhibition of neutral loss of phosphoric acid by N-terminal alcohols, implies that N-terminally catalyzed neutral loss proceeds through the direct loss of H 3 PO 4, rather than combined loss of water and HPO 3 , in accordance with the observation that the combined loss becomes less prevalent as charge mobility increases [17]. …”

“…To address this discrepancy, formation of a stable hydrogen bonded structure between the phosphate and a protonated basic residue, usually arginine, in place of a mobile proton, has been proposed for conferring electropositive character to the phosphate when mobile protons are not present. These groups have also found that, despite the uncommonness of sole loss of HPO 3 , there is a significant competing pathway in which HPO 3 is lost from the phosphosite with concurrent or sequential loss of water from elsewhere in the peptide [15, 17, 18]. …”

Detection of a phosphorylated glycine-serine linker in an IgG-based fusion protein

“…The neutral loss involves 2 competing cleavages of the phosphoester bond resulting in product ions with different structures but identical m/z values. First, there is a direct loss of H 3 PO 4 (98 Da) from the phosphorylated residue, secondly there is a combined loss of HPO 3 (80 Da) and H 2 O (18 Da) from the phosphorylation site and from an additional site within the peptide, respectively 35 . Indeed, the CID-MS/MS spectrum of the modified tryptic GS-linker peptide included an intense precursor ion signal with a −98 Da loss (pre(−98) + , m/z 1664.8) (Fig.…”