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Simply Extending the Mass Range in Electron Transfer Higher Energy Collisional Dissociation Increases Confidence in N-Glycopeptide Identification.

ABSTRACT: Glycopeptide-centric mass spectrometry has become a popular approach for studying protein glycosylation. However, current approaches still utilize fragmentation schemes and ranges originally optimized and intended for the analysis of typically much smaller unmodified tryptic peptides. Here, we show that by merely increasing the tandem mass spectrometry m/z range from 2000 to 4000 during electron transfer higher energy collisional dissociation (EThcD) fragmentation, a wealth of highly informative c and z ion fragment ions are additionally detected, facilitating improved identification of glycopeptides. We demonstrate the benefit of this extended mass range on various classes of glycopeptides containing phosphorylated, fucosylated, and/or sialylated N-glycans. We conclude that the current software solutions for glycopeptide identification also require further improvements to realize the full potential of extended mass range glycoproteomics. To stimulate further developments, we provide data sets containing all classes of glycopeptides (high mannose, hybrid, and complex) measured with standard (2000) and extended (4000) m/z range that can be used as test cases for future development of software solutions enhancing automated glycopeptide analysis.

SUBMITTER: Caval T

PROVIDER: S-EPMC6706795 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Simply Extending the Mass Range in Electron Transfer Higher Energy Collisional Dissociation Increases Confidence in N-Glycopeptide Identification.

Čaval Tomislav T Zhu Jing J Heck Albert J R AJR

Analytical chemistry 20190731 16

Glycopeptide-centric mass spectrometry has become a popular approach for studying protein glycosylation. However, current approaches still utilize fragmentation schemes and ranges originally optimized and intended for the analysis of typically much smaller unmodified tryptic peptides. Here, we show that by merely increasing the tandem mass spectrometry <i>m</i>/<i>z</i> range from 2000 to 4000 during electron transfer higher energy collisional dissociation (EThcD) fragmentation, a wealth of high ...[more]

PMID: 31287300

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Project description:Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E)XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or ?-elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation (CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simultaneous elucidation of glycan structures and peptide sequence.

Dataset Information

Simply Extending the Mass Range in Electron Transfer Higher Energy Collisional Dissociation Increases Confidence in N-Glycopeptide Identification.

Publications

Simply Extending the Mass Range in Electron Transfer Higher Energy Collisional Dissociation Increases Confidence in N-Glycopeptide Identification.

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