Formation and dissociation of phosphorylated peptide radical cations

Abstract

In this study, we generated phosphoserine- and phosphothreonine-containing peptide radical cations through low-energy collision-induced dissociation (CID) of the ternary metal-ligand phosphorylated peptide complexes [Cu(II)(terpy)(p)M](·2+) and [Co(III)(salen)(p)M](·+) [(p)M: phosphorylated angiotensin III derivative; terpy: 2,2':6',2''-terpyridine; salen: N,N'-ethylenebis(salicylideneiminato)]. Subsequent CID of the phosphorylated peptide radical cations ((p)M(·+)) revealed fascinating gas-phase radical chemistry, yielding (1) charge-directed b- and y-type product ions, (2) radical-driven product ions through cleavages of peptide backbones and side chains, and (3) different degrees of formation of [M - H(3)PO(4)](·+) species through phosphate ester bond cleavage. The CID spectra of the (p)M(·+) species and their non-phosphorylated analogues featured fragment ions of similar sequence, suggesting that the phosphoryl group did not play a significant role in the fragmentation of the peptide backbone or side chain. The extent of neutral H(3)PO(4) loss was influenced by the peptide sequence and the initial sites of the charge and radical. A preliminary density functional theory study, at the B3LYP 6-311++G(d,p) level of theory, of the neutral loss of H(3)PO(4) from a prototypical model--N-acetylphosphorylserine methylamide--revealed several factors governing the elimination of neutral phosphoryl groups through charge- and radical-induced mechanisms.

Figure 1

CID spectra of (a) [⁶³Cu^II(terpy)_pM]^·2+ and (b) [⁶⁵Cu^II(terpy)_pM]^·2+ (_pM = R_pSYIHPF)

Scheme 1

Collision-induced dissociation pathway of [Cu^II(terpy)_pM]^·2+ complex, where _pM = phosphoserine or phosphothreonine peptide

Figure 2

CID spectra of (a) [R_pSYIHPF]^·+ (ions in common with RSYIHPF are marked in red) and (b) [RGL_pSYG]^·+, the asterisk (*) denotes the loss of H₃PO₄

Figure 3

CID spectra of (a) [RSYIHPF]^·+ and (b) [RGLSYG]^·+

Figure 4

CID spectra of (a) [GG_pSY]^·+, (b) [G^·G_pSY]⁺, (c) [GG_pSW]^·+, and (d) [G^·G_pSW]⁺

Figure 5

Potential energy surfaces for the H₃PO₄ loss of N-acetylphosphorylserine methylamide analogues (a) cation through charge-driven pathways, (b) radical through radical-driven pathways, and (c) radical cation through charge-directed (in blue) and radical-driven (in black) pathway. The relative enthalpies at 0K at B3LYP/6-311++G(d,p) level are shown in kcal mol^–1