Characterization of intact N- and O-linked glycopeptides using higher energy collisional dissociation

Abstract

Simultaneous elucidation of the glycan structure and the glycosylation site are needed to reveal the biological function of protein glycosylation. In this study, we employed a recent type of fragmentation termed higher energy collisional dissociation (HCD) to examine fragmentation patterns of intact glycopeptides generated from a mixture of standard glycosylated proteins. The normalized collisional energy (NCE) value for HCD was varied from 30 to 60% to evaluate the optimal conditions for the fragmentation of peptide backbones and glycoconjugates. Our results indicated that HCD with lower NCE values preferentially fragmented the sugar chains attached to the peptides to generate a ladder of neutral loss of monosaccharides, thereby enabling the putative glycan structure characterization. In addition, detection of the oxonium ions enabled unambiguous differentiation of glycopeptides from non-glycopeptides. In contrast, HCD with higher NCE values preferentially fragmented the peptide backbone and, thus, provided information needed for confident peptide identification. We evaluated the HCD approach with alternating NCE parameters for confident characterization of intact N- and O-linked glycopeptides in a single liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. In addition, we applied a novel data analysis pipeline, so-called GlycoFinder, to form a basis for automated data analysis. Overall, 38 unique intact glycopeptides corresponding to eight glycosylation sites (six N-linked and two O-linked sites) were confidently identified from a standard protein mixture. This approach provided concurrent characterization of both the peptide and the glycan, thereby enabling comprehensive structural characterization of glycoproteins in a single LC–MS/MS analysis.

Data-independent oxonium ion profiling of multi-glycosylated biotherapeutics

“…These ions have been utilized in numerous advantageous approaches to qualitatively and quantitatively assess site-specific glycosylation. 16,17,23–28 However, oxonium ions have yet to be exploited to create agnostic profiles for high-throughput glycopeptide screening of multi-glycosylated therapeutics. Even with the substantial advancements made in glycoprotein analysis, it still remains difficult to unambiguously assign all potential sites of glycosylation and their associated glycans for complicated multi-glycosylated proteins.…”

Intact glycopeptide characterization using mass spectrometry

“…Second, the commonly used collisional based MS fragmentation method like collision-induced dissociation (CID) preferentially dissociates glycan while produces little peptide backbone fragments, which makes identification of peptide sequences difficult. In addition, the low m/z glycan oxonium ions, which are signature ions of glycopeptides[16, 17], may not be detected in the CID MS/MS spectra in ion-trap type MS. Therefore, an alternate fragmentation method is crucial for producing peptide backbone fragments.…”

“…Additionally, detailed glycan composition and structure information can be obtained separately [25, 26]; however, this information cannot be directly connected to specific glycosylation sites. An intact glycopeptide strategy (Figure 1) is emerging with encouraging results as an alternative to deglycosylation[17, 27, 28]. This new strategy keeps the glycan attached to its amino acids, which enables to elucidate the glycopeptide sequence, the glycosylation site, as well as the glycan composition simultaneously.…”

Engaging Challenges in Glycoproteomics: Recent Advances in MS-Based Glycopeptide Analysis

“…Other studies have made similar use of the energy- Glycoproteomics: recent advances in MS-based glycopeptide analysis Review resolved dissociation channels of glycopeptide ions [128][129][130][131]. Thus, CID is likely to gain broader use as a means of accessing peptide sequence ions in addition to the more frequently noted glycan fragmentation products.…”