PXD027536

PXD027536 is an original dataset announced via ProteomeXchange.

Title	A Linkage- Specific Sialic Acid Labeling Strategy Reveals Different Site-Specific Glycosylation Patterns in SARS-CoV-2 Spike Protein Produced in CHO and HEK Cell Substrates
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.
HostingRepository	PRIDE
AnnounceDate	2022-02-17
AnnouncementXML	Submission_2022-02-17_05:44:59.172.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Qiong Wang
SpeciesList	scientific name: Cricetulus barabensis; NCBI TaxID: 329629; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	amidated residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2021-07-25 14:43:40	ID requested
⏵ 1	2022-02-17 05:45:00	announced

Wang Q, Wang Y, Yang S, Lin C, Aliyu L, Chen Y, Parsons L, Tian Y, Jia H, Pekosz A, Betenbaugh MJ, Cipollo JF, A Linkage-specific Sialic Acid Labeling Strategy Reveals Different Site-specific Glycosylation Patterns in SARS-CoV-2 Spike Protein Produced in CHO and HEK Cell Substrates. Front Chem, 9():735558(2021) [pubmed]

ProteomeXchange project tag: Sars-cov-2, Covid-19

submitter keyword: Recombinant SARS-2 Spike, Glycopeptide, LC-MSMS, Sialic acids labeling

John F. Cipollo
contact affiliation	Center for Biologics Evaluation and Research (CBER), U.S. Food and Drug Administration (FDA)
contact email	John.Cipollo@fda.hhs.gov
lab head
Qiong Wang
contact affiliation	Johns Hopkins Universtiy
contact email	QiongWang314@gmail.com
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/02/PXD027536

PRIDE project URI

• PRIDE
  1. PXD027536
     1. Label: PRIDE project
     2. Name: A Linkage- Specific Sialic Acid Labeling Strategy Reveals Different Site-Specific Glycosylation Patterns in SARS-CoV-2 Spike Protein Produced in CHO and HEK Cell Substrates