Efficient HCD-pd-EThcD approach for N-glycan mapping of therapeutic antibodies at intact glycopeptide level

Li, Menglin; Zhu, Wenwen; Zheng, Hao; Zhang, Jinlan

doi:10.1016/j.aca.2021.339232

Cited by 7 publications

(5 citation statements)

References 30 publications

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“…Identification of glycopeptides used high‐resolution MS on an OT instrument to detect precursor ions, and those above an intensity threshold were selected for HCD FTMS/MS scans. This relatively energetic fragmentation yields abundant b‐ and y‐ions for spectral matching to peptide sequences, but can also produce oxonium ions (such as HexNAc 204.0867 m / z , HexNAc fragment 138.0545 m / z , and Hex(2)NAc 366.1396 m / z ) diagnostic for the presence of complex glycosylation of the precursor peptide 33,40,44,45 . The presence of these signature oxonium ions triggered data‐dependent EThcD FTMS/MS of a fresh precursor ion packet, yielding peptide fragments retaining one or more hexose groups or intact glycan, ideally confirming the glycosylation sequon's location 46 …”

Section: Resultsmentioning

confidence: 99%

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS‐CoV‐2 spike glycoprotein

Haynes,

Keppel,

Mekonnen

et al. 2024

Rapid Comm Mass Spectrometry

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RationaleHydrogen/deuterium exchange mass spectrometry (HDX‐MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat‐denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX‐MS, but one challenge for complete sequence coverage is N‐glycosylation sites. The deuteration of peptides post‐translationally modified by asparagine‐bound glycans (glycopeptides) has not always been identified in previous reports of HDX‐MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein.MethodsWe detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data‐dependent acquisition. An MS scan was used to identify precursor ions; if high‐energy collision‐induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low‐energy collision‐induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX‐MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2O.ResultsWe report HDX‐MS analysis of the SARS‐CoV‐2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N‐glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements.ConclusionInclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.

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Section: Resultsmentioning

confidence: 99%

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS‐CoV‐2 spike glycoprotein

Haynes,

Keppel,

Mekonnen

et al. 2024

Rapid Comm Mass Spectrometry

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“…Identification of glycopeptides used Fourier-transform mass spectrometry (FTMS) scans to detect precursor ions, and those above an intensity threshold were selected for high-energy collision-induced dissociation (HCD) FTMS/MS scans. This relatively energetic fragmentation yields abundant b-and y-ions for spectral matching to peptide sequences, but can also produce oxonium ions (such as HexNAc 204.0867 m/z , HexNAc fragment 138.0545 m/z , and Hex(2)NAc 366.1396 m/z ) diagnostic for the presence of complex glycosylation of the precursor peptide (33,39,43,44). The presence of these signature oxonium ions triggered data-dependent electron transfer low-energy collision-induced dissociation (EThcD) FTMS/MS of a fresh precursor ion packet, yielding peptide fragments retaining 1 or more hexose groups or intact glycan, ideally confirming the glycosylation sequon's location (45).…”

Section: Resultsmentioning

confidence: 99%

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike glycoprotein

Haynes,

Keppel,

Mekonnen

et al. 2023

Preprint

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Rationale. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein’s conformational dynamics across varied states, such as heat-denatured vs. native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. Methods. We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions, if high-energy collision-induced dissociation (HCD) MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans then electron transfer low-energy collision-induced dissociation (EThcD) MS/MS scans of the precursor identified the modified asparagine residue and the glycan’s mass. As in traditional HDX-MS the identified glycopeptides were then analyzed at the MS level in samples labeled with D O. Results. We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric pre-fusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural re-arrangements. Conclusion. Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.

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“…Careful selection of MS fragmentation schemes is required for informative MS/MS spectra for glycopeptide detection and characterization. While several studies have been performed to compare the use of different fragmentation mechanisms and energies [62–66], most of these studies have been performed on tryptic peptides, which fragment differently than endogenous peptides due to the different properties such as an increase in number of basic residues. The number of O‐linked and N‐linked neuropeptide glycoforms observed through sceHCD and pd‐EThcD fragmentation are compared in Figure 2A.…”

Section: Resultsmentioning

confidence: 99%

Enrichment and fragmentation approaches for enhanced detection and characterization of endogenous glycosylated neuropeptides

Phetsanthad

Roycroft

2022

Proteomics

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Glycosylated neuropeptides were recently discovered in crustaceans, a model organism with a well-characterized neuroendocrine system. Several workflows exist to characterize enzymatically digested peptides; however, the unique properties of endogenous neuropeptides require methods to be re-evaluated. We investigate the use of hydrophilic interaction liquid chromatography (HILIC) enrichment and different fragmentation methods to further probe the expression of glycosylated neuropeptides in Callinectes sapidus. During the evaluation of HILIC, we observed the necessity of a less aqueous solvent for endogenous peptide samples. This modification enabled the number of detected neuropeptide glycoforms to increase almost two-fold, from 18 to 36. Product ion-triggered electron-transfer/higher-energy collision dissociation enabled the site-specific detection of 55 intact N-and O-linked glycoforms, while the faster stepped collision energy higher-energy collisional dissociation resulted in detection of 25. Additionally, applying this workflow to five neuronal tissues enabled the characterization of 36 more glycoforms of known neuropeptides and 11 more glycoforms of nine putative novel neuropeptides. Overall, the database of glycosylated neuropeptides in crustaceans was largely expanded from 18 to 136 glycoforms of 40 neuropeptides from 10 neuropeptide families. Both macro-and micro-heterogeneity were observed, demonstrating the chemical diversity of this simple invertebrate, establishing a framework to use crustacean to probe modulatory effects of glycosylation on neuropeptides.

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Efficient HCD-pd-EThcD approach for N-glycan mapping of therapeutic antibodies at intact glycopeptide level

Cited by 7 publications

References 30 publications

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS‐CoV‐2 spike glycoprotein

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS‐CoV‐2 spike glycoprotein

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike glycoprotein

Enrichment and fragmentation approaches for enhanced detection and characterization of endogenous glycosylated neuropeptides

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