Assignment of Saccharide Identities through Analysis of Oxonium Ion Fragmentation Profiles in LC–MS/MS of Glycopeptides

Abstract

Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure-function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC-MS/MS-based glycoproteomic studies.

Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus

“…We have previously estimated that simple core-2 structures constitute ϳ10 -15% of total O-glycans in HEL fibroblasts (31). It is noteworthy to mention that in the present study we have identified a small fraction (ϳ6%) of the virus-derived glycosites potentially carrying both core-2 and core-1 O-glycans on the same sites in the peanut agglutinin-enriched peptides (87). The finding suggests that despite the presence of more complex O-glycans at certain sites, we might still detect them as biosynthetic intermediates.…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…The GlcNAc-derived fragments m/z 138.0545 and m/z 204.0867 are particularly useful diagnostic ions for such acquisition styles. In fact, the same oxonium ions are often generated upon beam-type CID (HCD) fragmentation of GalNAc-containing peptides, albeit in different intensities (123), meaning that these acquisition strategies are also useful for O-glycopeptides. The saccharide oxonium ion profile derived from glycopeptide fragmentation can even provide valuable supporting information of the monosaccharide compositions of the attached N-glycans (123,124).…”

“…In fact, the same oxonium ions are often generated upon beam-type CID (HCD) fragmentation of GalNAc-containing peptides, albeit in different intensities (123), meaning that these acquisition strategies are also useful for O-glycopeptides. The saccharide oxonium ion profile derived from glycopeptide fragmentation can even provide valuable supporting information of the monosaccharide compositions of the attached N-glycans (123,124). The original product-dependent (PD) method, which triggered ETD events upon recognition of m/z 204.086 in HCD-MS/MS (125), was lately followed up with a more informative approach where alternating ion trap CID and ETD events are triggered upon oxonium ion recognition in HCD-MS/MS spectra (126).…”

Structural characterisation of glycosylation of paramyxovirus attachment and fusion proteins by mass spectrometry

“…When analysed using high resolution and mass accuracy, the oxonium ions produced from the glycan portion of the glycopeptide can be used to identify the components of the glycan (203,218,219). In some cases, the relative abundance ratio of oxonium derived from HexNAc can indicate the presence of certain linkages of monosaccharides (GlcNAc or GalNAc) in a glycan (220)(221)(222).…”

“…The list of the oxonium ions is stored in an Excel spreadsheet within the data folder of OxoExtract which can be modified by the user. The oxonium ions used in this work were observed in samples, theoretical or were defined in the literature (219,220,238,251,(267)(268)(269) and have been represented in Table 3 …”