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 [CuII(terpy)pM]·2+ and [CoIII(salen)pM]·+ [pM: phosphorylated angiotensin III derivative; terpy: 2,2':6',2''-terpyridine; salen: N,N '-ethylenebis(salicylideneiminato)]. Subsequent CID of the phosphorylated peptide radical cations (pM·+) 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 – H3PO4]·+ species through phosphate ester bond cleavage. The CID spectra of the pM·+ 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 H3PO4 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 H3PO4 from a prototypical model—N-acetylphosphorylserine methylamide—revealed several factors governing the elimination of neutral phosphoryl groups through charge- and radical-induced mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-012-0479-7) contains supplementary material, which is available to authorized users.
Highlights
Protein post-translational modifications (PTMs) are essential processes in the regulation of cellular events because they are necessary steps toward rendering the functionality of a protein [1]
We first investigated the generation of phosphopeptide radical cations through collision-induced dissociation (CID) of copper(II)–ligand– peptide complexes
The spectrum features abundant signals at m/z 998.2, assigned to pM·+, and m/z 296.1, assigned to the complementary reduced product ion [63CuI(L)]+. We confirmed these spectral assignments by comparing the CID spectra of [63CuII(L)(pM)]·2+ with that of its isotope analogue [65CuII(L)(pM)]·2+ (Figure 1b)
Summary
Protein post-translational modifications (PTMs) are essential processes in the regulation of cellular events because they are necessary steps toward rendering the functionality of a protein [1]. Phosphorylation is a common PTM; in many proteins, the hydroxyl (–OH) group of the side chain of a tyrosine, serine, or threonine residue is. Mass spectrometry (MS)-based identification of phosphopeptides involves analysis of fragmentation patterns to determine the peptide sequence and the number and exact positions of the phosphorylation sites. Despite recent advances in MS-based proteomics, determining the exact modification sites of phosphorylated peptides remains challenging [3,4,5,6], partly because deciphering phosphopeptide sequences from fragmentation patterns requires an Received: 3 July 2012 Revised: 14 August 2012 Accepted: 14 August 2012 Published online: 12 September 2012
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