Dataset Information

Precision Characterization of Site-specific O-Acetylated Sialic Acids on N-Glycoproteins

ABSTRACT: O-Acetylation is a common modification of sialic acid, playing a significant role in glycoprotein stability, immune response, and cell development. The lack of efficient methods for direct analysis of O-acetylated sialoglycopeptides (O-AcSGPs) made the majority of identified O-acetylated sialic acids (O-AcSia) till now had no glycosite/glycoprotein information. Herein, we introduced a new workflow for precise interpretation of O-AcSGPs with probability estimation by recognizing the characteristic B and Y ions of O-AcSias.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Blood Serum

SUBMITTER: Jiechen Shen

LAB HEAD: Shisheng Sun

PROVIDER: PXD037161 | Pride | 2023-01-13

REPOSITORIES: Pride

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			Action	DRS
	ShenJ_MouseSerum_O-AcSGP_control_CE20_33.raw	Raw
	ShenJ_MouseSerum_O-AcSGP_control_CE20_33_result.xlsx	Xlsx
	ShenJ_MouseSerum_O-AcSGP_optimized_MIT_100ms_CE20_33.raw	Raw
	ShenJ_MouseSerum_O-AcSGP_optimized_MIT_100ms_CE20_33_result.xlsx	Xlsx
	ShenJ_MouseSerum_O-AcSGP_optimized_MIT_1s_CE20_33.raw	Raw

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Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes an extensively glycosylated spike protein that protrudes from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the predominant recombinant spike protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, recombinant full-size spike protein was produced transiently in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on spike glycans, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on glycans and glycopeptides from spike protein. We determined global and site-specific N-linked glycosylation patterns in soluble SARS-CoV-2 spike using MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation. We identified the glycan compositions at 21 and 19 out of the 22 predicted N-glycosylation sites of the SARS-CoV-2 spike proteins produced in CHO and HEK, respectively. The N-glycan site at 1158 position (N1158) and the N-glycan sites at 122 ,282 and 1158 positions (N122, N282 and N1158) were found to be unoccupied on spike secreted from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human spike proteins revealed that CHO-Spike presented more complex and higher sialylation (α2,3-linked) content while HEK-Spike displayed more high-mannose and minor content of α2,3- and α2,6-linked sialic acids. The N74 site represents the most heavily N-glycosylated site on both spike proteins while some high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801 and N1134) on HEK-Spike may differentially shield the virus compared to CHO-spike and provide a different host immune system interaction strategy. Collectively, these data underscore the importance of characterizing site-specific glycosylation on recombinant human spike protein from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in diagnostics and vaccines.

Dataset Information

Precision Characterization of Site-specific O-Acetylated Sialic Acids on N-Glycoproteins

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