The combination of electron capture dissociation and fixed charge derivatization increases sequence coverage for O-glycosylated and O-phosphorylated peptides

Abstract

Electron capture dissociation (ECD) has become an alternative method to collision-activated dissociation (CAD) to avoid gas-phase cleavage of post-translational modifications carried by side chains from the peptide backbone. Nonetheless, as illustrated herein by the study of O-glycosylated and O-phosphorylated peptides, the extent of ECD fragmentations may be insufficient to cover the entire peptide sequence and to localize accurately these modifications. The present work demonstrates that the derivatization of peptides at their N-terminus by a phosphonium group improves dramatically and systematically the sequence coverage deduced from the ECD spectrum for both O-glycosylated and O-phosphorylated peptides compared with their native counterparts. The exclusive presence of N-terminal fragments (c-type ions) in the ECD spectra of doubly charged molecular cations simplifies peptide sequence interpretation. Thus, the combination of ECD and fixed charge derivatization appears as an efficient analytical tool for the extensive sequencing of peptides bearing labile groups. (J Am Soc Mass Spectrom 2007, 18, 1405-1413) © 2007 American Society for Mass Spectrometry T he field of proteomics usually aims at differential characterization of the complete pattern of protein expression, for instance between cells in a normal state and those in a pathological state. Protein identification is now routinely achieved on a large scale (tens or hundreds of proteins per day) by combining enzymatic digestion, mass spectrometric analysis of the resulting peptides, and subsequent database search. If few peptides or short peptide sequences are sufficient for protein identifications into databases, a complete determination of the primary structure is nevertheless useful to detect modifications that can be involved in the regulation of protein function. Indeed, the catalytic property, the conformation, the turnover, or the subcellular localization of proteins are largely under the control of various peptide sequence processings named posttranslational modifications. These modifications (PTM) involve the proteolytic maturation of the proteins and/or the transfer of chemical groups (phosphorylation, glycosylation, acetylation, sumoylation) on specific amino-acids (serine, threonine, or tyrosine for O-phosphorylations, serine or threonine for Oglycosylations, asparagine for N-glycosylations). The most obvious strategy for mapping the modifications along the peptide chain involves a comparison between the sequence obtained by mass spectrometric data and the one deduced from the open reading frame coded by the gene sequence. However, a drawback arises from the propensity of groups conjugated to the amino acid side chains to dissociate readily upon collisional activation. For instance, collisionally-activated dissociation (CAD) MS/MS spectra of protonated precursor ions of phosphorylated peptides exhibit intense ions originating from the elimination of metaphosphoric (HPO 3 , loss of 80 Da) or orthophosphoric acid (H 3 PO 4 , loss of 98 Da) [1]. I...

Formation of Peptide Radical Cations (M+·) in Electron Capture Dissociation of Peptides Adducted with Group IIB Metal Ions

“…An important experimental parameter is the nature of the charge carriers. Several researchers have studied the impact of replacing the mobile protons with fixed charges using functionalities such as quaternary amino groups [20][21][22][23]. Our group and other researchers have focused on the use of divalent and trivalent metal ions as charge carriers [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38].…”

Electron capture in charge-tagged peptides. Evidence for the role of excited electronic states

“…As a chargecarrying group we chose tris-(2,4,6-trimethoxyphenyl)-phosphonium (TMPP) that can be selectively attached to amino groups at the peptide N-terminus or in the lysine side-chain via a methylenecarboxamide linker [16]. Analogous TMPP derivatives have been used to introduce a fixed charge in larger peptides for ion dissociation [17] and ECD studies [18]. The other charge needed for ECD is introduced as a proton by electrospray ionization.…”

Enhanced Electron Transfer Dissociation of Peptides Modified at C-terminus with Fixed Charges

“…O'Connor et al labeled the lysine side chains of peptides with 2,4,6-trimethylpyridinium groups, and the labeled peptides exhibited fewer backbone cleavages and more prominent side-chain cleavages upon ECD [42]. Chamot-Rooke et al derivatized the N-termini of peptides with phosphonium groups to improve the sequence coverage obtained upon ECD of O-glycosylated and Ophosphorylated peptides [43]. The Chait group pursued the derivatization of cysteines with N,N-dimethyl-2-chloro-ethylamine (DML), resulting in incorporation of very basic amine groups and ultimately yielding better sequence coverage upon subsequent ETD [44].…”