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.

Keywords: Automated identification; Glycopeptides; Glycosylation; HCD; LC–MS/MS; NCE.

Fig. 1

Schematic representation of the experimental workflow.

Fig. 2

Examples of N-linked glycopeptide (A,B) and O-linked glycopeptide (C,D) tandem mass spectra acquired using HCD collisional energy: NCE = 30% (A,C) and NCE = 50% (B,D). B ions and Y ions (uppercase) are glycan fragmentation ions, and the designations follow the rule described in the previous publication [30]; b ions and y ions (lowercase) are peptide fragmentation ions. Monosaccharides are labeled according to the nomenclature outlined by the Consortium for Functional Glycomics (http://glycomics.scripps.edu/CFGnomenclature.pdf); squares, circles, and diamonds represent HexNAc, Hex, and NeuAc, respectively. Glycan is represented by the total number of each type of monosaccharide (i.e., 2* □ represents two HexNAc in the detected glycan). Mass-to-charge ratio (m/z) and charge state (CS) of the selected precursor ion are listed on the top of each spectrum.

Fig. 3

Normalized intensity of N- and O-linked glycopeptide fragment ions plotted for different HCD collisional energies: (A) peptide plus monosaccharide ions for N-linked glycopeptides; (B) peptide plus glycopeptide ions for O-linked glycopeptides; (C) singly charged peptide and y ions derived from the N-linked glycopeptides; (D) doubly charged peptide and y ions corresponding to the O-linked glycopeptide.

Fig. 4

Identification of N-linked glycopeptide from A. niger secretome (AMYG_ASPNG glucoamylase) using HCD with alternating NCE parameters: (A) HCD analysis under low NCE settings; (B) HCD analysis under high NCE settings.