Dataset Information

Optimization of O-GIG for O-glycopeptide Characterization with Sialic Acid Linkage Determination -Figure 2

ABSTRACT: O-glycoprotein analysis has been historically challenging due, in part, to a dearth of available enzymes active in release of O-glycans. Moreover, chemical releasing methods, such as β-elimination/Michael addition are not specific to O-glycan release and can also eliminate phosphoryl substitutions. Both of these events leave behind deaminated serine and threonine and thus can lead to ambiguous structural conclusions. Recently, the O-protease OpeRATOR, derived from intestinal bacteria and expressed in E. coli, has become commercially available. Digestion of O-glycoprotein yields O-glycopeptides cleaved at the N-terminal end of serine/threonine with O-glycan remaining intact. The enzyme has broad substrate specificity and includes mammalian Cores 1-8. However, OpeRATOR is not fully active toward sialylated glycoproteins and therefore it is suggested that this acidic residue be removed prior to digestion, thus, sacrificing structural information. In this study, we investigated the performance of OpeRATOR under a range of conditions, including buffer selection, pH, sialic acid modification, and digestion temperature in order to optimize enzymatic activity with special emphasis on sialylated glycosites. Conditions derived in this work facilitate OpeRATOR digestion of fully sialylated O-glycopeptides mass tagged to identify the sialyl linkage, thus, facilitating analysis of these charged O-glycopeptides, which are often important in biological processes.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Bos Taurus (bovine)

SUBMITTER: LIsa Parsons

LAB HEAD: John F. Cipollo

PROVIDER: PXD020039 | Pride | 2020-07-28

REPOSITORIES: Pride

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Optimization of O-GIG for O-Glycopeptide Characterization with Sialic Acid Linkage Determination.

Yang Shuang S Wu Wells W WW Shen Rongfong R Sjogren Jonathan J Parsons Lisa L Cipollo John F JF

Analytical chemistry 20200728 16

O-Glycoprotein analysis has been historically challenging due, in part, to a dearth of available enzymes active in the release of O-glycans. Moreover, chemical releasing methods, such as β-elimination/Michael addition, are not specific to O-glycan release and can also eliminate phosphoryl substitutions. Both of these events leave behind deaminated serine and threonine and thus can lead to ambiguous structural conclusions. Recently, the O-protease OpeRATOR, derived fro ...[more]

PMID: 32663021

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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.

Project description:Recombinant proteins are of great interest in glycobiology and proteomics, known especially for their reproducibility and accessibility. However, variation in glycosylation among recombinant glycoproteins is not well understood and may depend on numerous conditions in the biomanufacturing process. In order to confidently assess variation in glycosylation measurements, it is vital to both optimize the measurement of, and determine the degree of variation between, distributions of glycosylation on specific sites of glycoproteins. This is especially important for glycoproteins that are known to have rapid sequence changes, such as with different influenza strains. In this study, eight strains of recombinant influenza hemagglutinin and neuraminidase produced from HEK293 cell line were obtained from four vendors and digestion was conducted using a series of complex multi-enzymatic methods designed to isolate glycopeptide sequons. Site-specific glycosylation profiles of intact glycopeptides were produced using mass spectrometric evaluation on an orbitrap system and compared using spectral similarity scores. Variation in glycan abundances and distribution was most pronounced between different strains of virus (similarity score = 383 out of 1000), whereas replicates resulted in low variation (similarity score = 957 out of 1000). Glycan variation was also measured based on differences between vendors, lots, batches, protease digestion, and intra-protein site. The most abundant glycans in all of these influenza glycoproteins were monofucosylated and complex, as reported by other laboratories. However, it was found that different vendors can produce very different glycan distributions for the same glycosylation site. Notably, it is demonstrated that glycan distributions are similar for conserved regions of influenza glycoproteins. Overall, these methods present a potential use in developing reproducible measurements of glycosylated biologics for quality control or making more informed decisions in biomanufacturing.

			Action	DRS
	EEDAOGP1.xlsx	Xlsx
	EEDAOGP2.xlsx	Xlsx
	EEDAOGP3.xlsx	Xlsx
	Fetuin_EEDA_rep1.raw	Raw
	Fetuin_EEDA_rep2.raw	Raw

Dataset Information

Optimization of O-GIG for O-glycopeptide Characterization with Sialic Acid Linkage Determination -Figure 2

Dataset's files

Publications

Optimization of <i>O-</i>GIG for <i>O</i>-Glycopeptide Characterization with Sialic Acid Linkage Determination.

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