Dataset Information

HEK-AXL glycopeptides supporting GlycopeptideGraphMS development

ABSTRACT: To support the development of a novel bioinformatic solution for glycopeptide identification, the cancer-associated AXL glycoprotein was used to validate the approach. AXL has 6 N-glycan sites.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Matthew Choo

LAB HEAD: Terry Nguyen-Khuong

PROVIDER: PXD009222 | Pride | 2019-05-23

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	02BYONICtriplexgraphms.csv	Csv
	03BYONICmergedinclistxgraphms.csv	Csv
	101a-HEK-AXL_500ng_OT_HCD30EThcD.raw	Raw
	102a-HEK-AXL_500ng_OT_HCD30EThcD.mzML	Mzml
	301-galglcnac_rep1.raw	Raw

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Publications

GlycopeptideGraphMS: Improved Glycopeptide Detection and Identification by Exploiting Graph Theoretical Patterns in Mass and Retention Time.

Choo Matthew S MS Wan Corrine C Rudd Pauline M PM Nguyen-Khuong Terry T

Analytical chemistry 20190523 11

The leading proteomic method for identifying N-glycosylated peptides is liquid chromatography coupled with tandem fragmentation mass spectrometry (LCMS/MS) followed by spectral matching of MS/MS fragment masses to a database of possible glycan and peptide combinations. Such database-dependent approaches come with challenges such as needing high-quality informative MS/MS spectra, ignoring unexpected glycan or peptide sequences, and making incorrect assignments because some glycan combinations are ...[more]

PMID: 31079452

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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.