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Sequential N/O-glycosylation analysis of heavily glycosylated HIV-1 gp120 by combination of electron-transfer/higher-energy collisional dissociation and stepped collision energy/higher-energy collisional dissociation (EThcD-sceHCD)

ABSTRACT: The envelope (Env) glycoprotein on the surface of human immunodeficiency virus type 1 (HIV-1) which decorated with a dense array of glycans is a determinant for viral invasion and host immune response of HIV-1 and a major target for a preventive HIV-1 vaccine. Improved vaccine design requires an understanding of the detailed information about the glycan type on each glycosite. Here, we used our well-established sequential glycoproteomic workflow to characterize the N/O-glycosylation of HIV-1 gp120 at the level of native intact glycopeptides based on a stepped collision energy/higher-energy collisional dissociation (sceHCD) mass spectrometry (sceHCD-MS/MS), and a combined electron transfer/higher-energy collisional dissociation (EThcD) and sceHCD mass spectrometry (EThcD-sceHCD-MS/MS).

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Cell Culture

SUBMITTER: Yong Zhang

LAB HEAD: Yong Zhang

PROVIDER: PXD025078 | Pride | 2021-11-03

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	EThcD-HCD.rar	Other
	HCD.rar	Other
	HIV1-gp120-Trypsin-GluC-after-enrichment-DeGP-R1.raw	Raw
	HIV1-gp120-Trypsin-GluC-after-enrichment-DeGP-R2.raw	Raw
	HIV1-gp120-Trypsin-GluC-after-enrichment-DeGP-R3.raw	Raw

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Optimal Dissociation Methods Differ for N- and O-glycopeptides

Project description:Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Furthermore, N-glycoproteomics has made significant progress in the last few years, and there is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and comment on their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.

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