Classification of Tandem Mass Spectra for Identification of N- and O-linked Glycopeptides

Abstract

Analysis of intact glycopeptides by mass spectrometry is essential to determining the microheterogeneity of protein glycosylation. Higher-energy collisional dissociation (HCD) fragmentation of glycopeptides generates mono- or disaccharide ions called oxonium ions that carry information about the structure of the fragmented glycans. Here, we investigated the link between glycan structures and the intensity of oxonium ions in the spectra of glycopeptides and utilized this information to improve the identification of glycopeptides in biological samples. Tandem spectra of glycopeptides from fetuin, glycophorin A, ovalbumin and gp120 tryptic digests were used to build a spectral database of N- and O-linked glycopeptides. Logistic regression was applied to this database to develop model to distinguish between the spectra of N- and O-linked glycopeptides. Remarkably, the developed model was found to reliably distinguish between the N- and O-linked glycopeptides using the spectral features of the oxonium ions using verification spectral set. Finally, the performance of the developed predictive model was evaluated in HILIC enriched glycopeptides extracted from human serum. The results showed that pre-classification of tandem spectra based on their glycosylation type improved the identification of N-linked glycopeptides. The developed model facilitates interpretation of tandem mass spectrometry data for assignment of glycopeptides.

Assignment of coronavirus spike protein site-specific glycosylation using GlycReSoft

“…Glycopeptide identification algorithms use peptide-centric, glycan-centric or complete approaches. The peptide-centric method focusses on identifying the peptide backbone sequence, may use peptide + Y ions, but do not control for the false discovery rate of the glycan [37]. By contrast, glycan-centric methods [38,39] identify the attached glycan but do not use peptide backbone dissociation to assign the peptide sequence.…”

“…As in proteomics, glycopeptide data are searched using target decoy analysis [58][59][60] whereby targets and decoys compete for spectral matching. Some published methods for glycopeptides use structural properties to optimize model performance [30,33,44] or employ hierarchical filters [26,37,47,49] to optimize results. For HCD, stepped collision energies most consistently produce peptide+Y ions and peptide bn and yn ions that characterize the glycopeptide glycan and peptide backbone independently [49,61,62].…”

Reanalysis of global proteomic and phosphoproteomic data identified a large number of glycopeptides

“…Finally, the GPQuest used in this study is a freely available software focused on the high-throughput intact glycopeptide analysis, utilizing HCD MS/MS spectra 37 . The software provides FDR estimation for each assignment of the identified glycopeptide, as well as predicting glycosylation types based on generated oxonium ions 38 .…”

Identification of Post-translational Modifications on Odorant-Binding Protein Isoforms from Pig Olfactory Secretome by High-Resolution Mass Spectrometry: O-β-N-acetylglucosaminylation and Phosphorylation

“…Indeed, the study of native proteins in complex mixture could explain why we did not find the oxonium ions (204.08) for HexNAcylation, typical of the HCD method (Zhao et al, 2011). These ions are usually detected in glycoprotein samples enriched in O-GlcNAcylated peptides (Toghi Eshghi et al, 2016) or exclusively composed of synthetic O-GlcNAcylated peptides (Chalkley and Burlingame, 2001;Malaker et al, 2017). In this study, identification of modified peptides was made by a manual assignment approach instead of using software as it is done in almost all PTM identification/localization papers.…”