Concurrent Automated Sequencing of the Glycan and Peptide Portions of O-Linked Glycopeptide Anions by Ultraviolet Photodissociation Mass Spectrometry

Abstract

O -glycopeptides are often acidic owing to the frequent occurrence of acidic saccharides in the glycan, rendering traditional proteomic workflows that rely on positive mode tandem mass spectrometry (MS/MS) less effective. In this report, we demonstrate the utility of negative mode ultraviolet photodissociation (UVPD) MS for the characterization of acidic O-linked glycopeptide anions. This method was evaluated for a series of singly- and multiply-deprotonated glycopeptides from the model glycoprotein kappa casein, resulting in production of both peptide and glycan product ions that afforded 100% sequence coverage of the peptide and glycan moieties from a single MS/MS event. The most abundant and frequent peptide sequence ions were a/x-type products, which, importantly, were found to retain the labile glycan modifications. The glycan-specific ions mainly arose from glycosidic bond cleavages (B, Y, C, and Z ions) in addition to some less common cross-ring cleavages. Based on the UVPD fragmentation patterns, an automated database searching strategy (based on the MassMatrix algorithm) was designed that is specific for the analysis of glycopeptide anions by UVPD. This algorithm was used to identify glycopeptides from mixtures of glycosylated and non-glycosylated peptides, sequence both glycan and peptide moieties simultaneously, and pinpoint the correct site(s) of glycosylation. This methodology was applied to uncover novel site-specificity of the O-linked glycosylated OmpA/MotB from the “superbug” A. baumannii to help aid in the elucidation of the functional role that protein glycosylation plays in pathogenesis.

Data-independent oxonium ion profiling of multi-glycosylated biotherapeutics

“…Advances in glycopeptide identification have certainly been made through the utilization of alternative MS/MS techniques such as electron transfer dissociation (ETD) 14–17 and ultraviolet photodissociation (UVPD) 18,19 to improve the fragmentation information generated through MS/MS. Furthermore, data-independent analysis (DIA) of glycopeptides has recently been applied to glycopeptide analysis to circumvent the traditional reliance on data-dependent acquisition (DDA) of LC-MS/MS data, 20–22 which relies on peak picking of the most abundant species from the MS1 spectra to initiate MS/MS, a process that can yield incomplete site-specific characterization.…”

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

“…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.…”

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

“…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. A number of researchers have noted that while CID and IRMPD tend to preferentially cleave the oligosaccharide moiety, ECD and ETD characteristically result in cleavage of only the polypeptide backbone.…”