Dataset Information

N- and O-Glycoproteomic Analysis of Complex Proteoglycans

ABSTRACT: Proteoglycans are a small but diverse family of proteins that play a wide variety of roles at the cell surface and extracellular matrix. In addition to their glycosaminoglycan (GAG) chains, they are also N- and O-glycosylated. All of these types of glycosylation are crucial to their function, but present a considerable analytical challenge. Using serial proteolysis and electron capture/higher energy collisional dissociation (EThcD) we seek to improve the sequence coverage of several complex proteoglycans. In many cases, the use of HCD alone allows for the identification of more glycopeptides, but EThcD allows for the confident assignment of glycan compositions on multiply glycosylated peptides. Additionally, glycoproteomics searches identify glycopeptides in otherwise poorly covered regions of proteoglycans. The development of these and other analytical tools may permit glycoproteomic similarity comparisons in biological samples.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

SUBMITTER: Margaret Downs

LAB HEAD: Joseph Zaia

PROVIDER: PXD041935 | Pride | 2025-05-06

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Aggpeptides_1_1_0.mzid.gz	Mzid
	AggrecanGlycopeptideReports.pdf	Pdf
	Aggrecan_GluC_B_HCD30_EThcD20.mgf	Mgf
	Aggrecan_GluC_B_HCD30_EThcD20.raw	Raw
	Aggrecan_GluC_B_HCDtrig.mgf	Mgf

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Publications

Analysis of complex proteoglycans using serial proteolysis and EThcD provides deep N- and O-glycoproteomic coverage.

Downs Margaret M Curran Jillian J Zaia Joseph J Sethi Manveen K MK

Analytical and bioanalytical chemistry 20230920 28

Proteoglycans are a small but diverse family of proteins that play a wide variety of roles at the cell surface and in the extracellular matrix. In addition to their glycosaminoglycan (GAG) chains, they are N- and O-glycosylated. All of these types of glycosylation are crucial to their function but present a considerable analytical challenge. We describe the combination of serial proteolysis followed by the application of higher-energy collisional dissociation (HCD) and electron transfer/higher-e ...[more]

PMID: 37728749

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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.