Fragmentation characteristics of glycopeptides

Abstract

Mass spectrometric analysis of glycopeptides is an emerging strategy for analysis of glycosylation patterns. Here we present an approach using energy resolved collision induced decomposition (CID) spectra to determine structural features of glycopeptides. Fragmentation of multiply protonated glycopeptides proceeds by a series of competing charge separation processes by cleavage of a glycosidic bond, each producing two charged products: a singly charged, "B" type sugar (oxonium) ion, and a complementary high mass fragment. Energy requirements (activation energies) of these processes are similar to each other, and are far less, than that required for peptide fragmentation. At higher collision energies these first generation products fragment further, yielding a complex fragmentation pattern. Analysis of low energy spectra (those corresponding to ca. 50% survival yield) are straightforward; the ions observed correspond to structural features present in the oligosaccharide, and are not complicated by consecutive reactions. This makes it feasible to identify and distinguish antenna-and core-fucosylated isomers; antenna fucosylation usually suggests presence of the Lewis-X antigen. In general, analysis of the triply protonated molecules are most advantageous, where neutral losses and monosaccharide oxonium ion formation are less abundant.

A Comparison of Energy-Resolved Vibrational Activation/Dissociation Characteristics of Protonated and Sodiated High Mannose N-Glycopeptides

“…The Y 1 glycan fragment (i.e., the peptide group with one remaining GlcNAc residue attached) was also observed, and by a significant margin constituted the most abundant fragment ion in this spectrum. This is consistent with previous observations that cleavage between the two GlcNAc residues of the N-linked glycan core is a favored process [14][15][16][17][18]. Under these conditions, the CID spectrum of the hybrid protonated sodium adduct (Figure 4b) was highly distinct from that of the doubly protonated precursor.…”

“…Substantial effort has been directed towards exploiting the specific advantages afforded by different ion dissociation methods for glycopeptide analysis, including those based on ion-neutral interactions, as in collision-induced dissociation (CID) [14][15][16][17][18]; ion-electron and ion-ion reactions, as in electron capture dissociation (ECD) and electron transfer dissociation (ETD) [19][20][21][22]; and irradiation with photons, as in infrared multiphoton dissociation (IRMPD) and ultraviolet photodissociation (UVPD) [23][24][25][26]. By comparison, less attention has been paid to the effects of charge carrier upon glycopeptide dissociation patterns and energetics.…”

Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

“…Nevertheless, little or no peptide sequence information is available when glycopeptides are subjected to CID. Recent efforts to increase the peptide backbone dissociation of glycopeptides in CID have been published (81,82): In these works, the collisional energy was modulated synergistically during the data acquisition period so that the energy-resolved CID spectrum would contain both glycan and peptide fragmentation (81,82). However, that method does not yet provide consistent backbone fragmentation across all different glycopeptides undergoing CID.…”

Energy-resolved collision-induced dissociation pathways of model N-linked glycopeptides: implications for capturing glycan connectivity and peptide sequence in a single experiment

“…22,23 The acquisition and subsequent interpretation of informative tandem mass spectrometry (MS/MS) data for glycopeptide ions stand among the most pressing of these challenges. Signicant effort has been made to maximize the information content of glycopeptide ion dissociation spectra, including those obtained through vibrational activation/dissociation methods such as collisioninduced dissociation (CID) 24,25 and infrared multiphoton dissociation (IRMPD); 26,27 ion-electron and ion-ion reactions resulting in electron capture dissociation (ECD) 28,29 or electron transfer dissociation (ETD); 30,31 and irradiation with ultraviolet photons in order to achieve ultraviolet photodissociation (UVPD). 32,33 One useful outcome of these investigations has been the observation of a high degree of complementarity between the vibrational activation/dissociation spectra and the electron capture/transfer dissociation spectra of glycopeptide ions.…”