Strategy Integrating Stepped Fragmentation and Glycan Diagnostic Ion-Based Spectrum Refinement for the Identification of Core Fucosylated Glycoproteome Using Mass Spectrometry

Abstract

Core fucosylation (CF) is a special glycosylation pattern of proteins that has a strong relationship with cancer. The Food and Drug Administration (FDA) has approved the core fucosylated α-fetoprotein as a biomarker for the early diagnosis of hepatocellular carcinoma (HCC). The technology for identifying core fucosylated proteins has significant practical value. The major method for core fucosylated glycoprotein/glycopeptide analysis is neutral loss-based MS(3) scanning under collision-induced dissociation (CID) by ion trap mass spectrometry. However, due to the limited speed and low resolution of the MS(3) scan mode, it is difficult to achieve high-throughput, with only dozens of core fucosylated proteins identified in a single run. In this work, we developed a novel strategy for the identification of CF glycopeptides at a large scale, integrating the stepped fragmentation function, one novel feature of quadrupole-orbitrap mass spectrometry, with "glycan diagnostic ion"-based spectrum optimization. By using stepped fragmentation, we were able to obtain both highly accurate glycan and peptide information of a simplified CF glycopeptide in one spectrum. Moreover, the spectrum could be recorded with the same high speed as the conventional MS(2) scan. By using the "glycan diagnostic ion"-based spectrum refinement method, the efficiency of the CF glycopeptide discovery was significantly improved. We demonstrated the feasibility and reproducibility of our method by analyzing CF glycoproteomes of mouse liver tissue and HeLa cell samples spiked with standard CF glycoprotein. In total, 1364 and 856 CF glycopeptides belonging to 702 and 449 CF glycoproteins were identified, respectively, within a 78-min gradient analysis, which was approximately a 7-fold increase in the identification efficiency of CF glycopeptides compared to the currently used method. In this work, we took core fucosylated glycopeptides as a practical example to demonstrate the great potential of our novel method for use in glycoproteome analysis, and we also anticipate using the flexible novel method in other research fields.

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

“…The Y 1 glycan fragment (i.e., the peptide group with one remaining GlcNAc residue attached) was also observed, and by a significant margin constituted the most abundant fragment ion in this spectrum. This is consistent with previous observations that cleavage between the two GlcNAc residues of the N-linked glycan core is a favored process [14][15][16][17][18]. Under these conditions, the CID spectrum of the hybrid protonated sodium adduct (Figure 4b) was highly distinct from that of the doubly protonated precursor.…”

“…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.…”

A biomimetic design for a sialylated, glycan-specific smart polymer

“…When the solvent polarity was improved to 70% acetonitrile/50 mM NH 4 FA, the bound glycopeptides dissociated from the material surfaces and were detected by MS. Figure 6c displays the MS of the fetuin tryptic digests mixed with 10-fold BSA digest interference after enrichment by our material. After comparing the results with the glycopeptide database and analyzing them by tandem MS/MS, 43 30 glycopeptide signals were identified, and among them, 23 glycopeptides possessed the typical structures of sialo-complex-type N-linked glycans. The other seven glycopeptides were equipped with a common pentasaccharide core.…”

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

“…Fragmentation can also result in the production of peptide b-and y-ions but factors such as peptide sequence, precursor m/z and the level of dissociation energy can affect sequence coverage (214,216). When employing collisional energies, a stepped method can be applied where a precursor is fragmented with multiple collision energies (typically low, medium and high) and the fragment ions are measured in one MS/MS spectrum.…”

“…Furthermore, the identification of Y1+dHex can indicate core fucosylation (146,216). The prescribed rules were translated into the OxoExtract Java application by Dr Christine Hoogland.…”