Gas-phase intermolecular phosphate transfer within a phosphohistidine phosphopeptide dimer - PubMed (original) (raw)

Gas-phase intermolecular phosphate transfer within a phosphohistidine phosphopeptide dimer

Maria-Belen Gonzalez-Sanchez et al. Int J Mass Spectrom. 2014.

Abstract

The hydrogen bonds and electrostatic interactions that form between the protonated side chain of a basic residue and the negatively charged phosphate of a phosphopeptide can play crucial roles in governing their dissociation pathways under low-energy collision-induced dissociation (CID). Understanding how phosphoramidate (i.e. phosphohistidine, phospholysine and phosphoarginine), rather than phosphomonoester-containing peptides behave during CID is paramount in investigation of these problematic species by tandem mass spectrometry. To this end, a synthetic peptide containing either phosphohistidine (pHis) or phospholysine (pLys) was analyzed by ESI-MS using a Paul-type ion trap (AmaZon, Bruker) and by traveling wave ion mobility-mass spectrometry (Synapt G2-S_i_, Waters). Analysis of the products of low-energy CID demonstrated formation of a doubly 'phosphorylated' product ion arising from intermolecular gas-phase phosphate transfer within a phosphopeptide dimer. The results are explained by the formation of a homodimeric phosphohistidine (pHis) peptide non-covalent complex (NCX), likely stabilized by the electrostatic interaction between the pHis phosphate group and the protonated _C_-terminal lysine residue of the peptide. To the best of our knowledge this is the first report of intermolecular gas-phase phosphate transfer from one phosphopeptide to another, leading to a doubly phosphorylated peptide product ion.

Keywords: CID; Gas-phase dimer; Histidine phosphorylation; NCX, non-covalent complex; Non-covalent interactions; Phosphoramidate; Phosphotransfer; TWIMS, travelling wave ion mobility-mass spectrometry; pArg, phosphoarginine; pHis, phosphohistidine; pLys, phospholysine.

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Figures

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Graphical abstract

Fig. 1

Fig. 1

ESI-ion trap full scan mass spectrum of the products of the reaction between peptide FVIAFILHLVK and potassium phosphoramidate (KNH2PO3H2). Inset shows an enhanced region of the mass spectrum encompassing m/z range 1280–1400.

Fig. 2

Fig. 2

CID product ion mass spectrum of the doubly charged ion of the phosphorylated peptide p[FVIAFILHLVK+2H]2+ at m/z 690.5, indicating a heterogeneous population of [FVIAFILpHLVK+2H]2+ and [FVIAFILHLVpK+2H]2+, whose specific y-ions are labeled in italics (gray). (Δ) Loss of 80 Da (HPO3); (*) Observation of both phosphorylated and non-phosphorylated product ions.

Fig. 3

Fig. 3

CID product ion mass spectrum of the singly charged ion of the phosphorylated peptide [p(FVIAFILHLVK)+H]+ at m/z 1379.5. (Δ) Loss of 80 Da (HPO3).

Fig. 4

Fig. 4

Traveling wave ion mobility-mass spectrometry analysis of the ions at m/z 1379.5 demonstrating a mixed population of singly protonated phosphopeptide monomer [M+H]+ and doubly protonated dimer [M2+2H]2+. (A) Isotopic distribution and (B) arrival time distribution (ATD) of the mixed population. Inset (A) depicts the theoretical isotope distribution (assuming 1:1 stoichiometry) of the [M+H]+ and [M2+2H]2+. Extracted ion current for the (C) longer (red) and (D) shorter (blue) ATDs (E) indicating mobility separation of the monomeric and dimeric populations.

Fig. 5

Fig. 5

MS3 CID product ion mass spectrum of ions at m/z 1459.4 generated by CID of ions at m/z 1379.5. The doubly ‘phosphorylated’ peptide is represented by p(FVIAFILpHLVK). (Δ) Loss of 80 Da (HPO3).

Scheme 1

Scheme 1

Proposed mechanism for the formation of the ions at m/z 1459.8. The scheme depicts a homodimer of the phosphopeptide FVIAFILpHLVK (m/z 1379.8), whose components can undergo elimination of metaphosphoric acid HPO3 and generation of a transient ternary complex, which then evolves to give the dephosphorylated peptide at m/z 1299.8 and the ‘doubly’ phosphorylated peptide at m/z 1459.8.

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