Dataset Information

Solid-phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) for comprehensive characterization of glycoproteins

ABSTRACT: A novel chemoenzymatic method termed solid phase extraction of N-linked Glycans And Glycosite-containing peptides (NGAG) for the simultaneous analysis of N-glycans, glycosite-containing peptides, and intact N-glycopeptides with site-specific glycosylation information.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Ovary

SUBMITTER: Paul Aiyetan

LAB HEAD: Hui Zhang

PROVIDER: PXD001571 | Pride | 2015-11-23

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Bovine_Fetuin.fasta	Fasta
	RefSeq.human.20130729.fasta	Fasta
	RefSeq.human.N_glycosite_to_U.20130729.fasta	Fasta
	SunS_10_OVCAR3_Control_with_SILAC_Global_F1.raw	Raw
	SunS_10_OVCAR3_Control_with_SILAC_Global_F10.raw	Raw

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Publications

Comprehensive analysis of protein glycosylation by solid-phase extraction of N-linked glycans and glycosite-containing peptides.

Sun Shisheng S Shah Punit P Eshghi Shadi Toghi ST Yang Weiming W Trikannad Namita N Yang Shuang S Chen Lijun L Aiyetan Paul P Höti Naseruddin N Zhang Zhen Z Chan Daniel W DW Zhang Hui H

Nature biotechnology 20151116 1

Comprehensive characterization of protein glycosylation is critical for understanding the structure and function of glycoproteins. However, due to the complexity and heterogeneity of glycoprotein conformations, current glycoprotein analyses focus mainly on either the de-glycosylated glycosylation site (glycosite)-containing peptides or the released glycans. Here, we describe a chemoenzymatic method called solid phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) for the ...[more]

PMID: 26571101

Publication: 1/2

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Project description:Proteolytic processing is an irreversible post-translational modification functioning as a ubiquitous regulator of cellular activity. Protease activity is tightly regulated by control of gene expression, compartmentalisation of enzyme and substrate, zymogen activation, enzyme inactivation, and substrate availability. Emerging evidence suggests that proteolysis can also be regulated by substrate glycosylation and that glycosylation of individual sites on a substrate can decrease, or in rare cases, increase its sensitivity to proteolysis. In the present study we investigated the relationship between site-specific O-glycosylation and proteolytic cleavage of extracellular proteins. By in silico analysis we found a significant association between O-glycosylation sites and cleavage sites. We then used a positional proteomic strategy, Terminal Amine Isotopic Labelling of Substrates (TAILS) to map in vivo cleavage sites in HepG2 cells with and without one of the key initiating GalNAc-transferases, GalNAc-T2. To reduce complexity, the comparison were performed in so-called SimpleCells. In these cells the Core 1 β3-galactosyltransferase-specific molecular chaperone (COSMC; C1GALT1C1) has been knocked out, resulting in global truncation of O-glycans leaving only the initial GalNAc. Surprisingly, we found that loss of GalNAc-T2 resulted in not only an increase in cleavage, but also a decrease in cleavage across a broad range of other substrates, including key regulators of the protease network. GALNT2 has previously been identified as a candidate gene for dyslipidaemia and has been shown to specifically glycosylate central regulators of lipid homeostasis, here we find altered processing of several of these regulators including Apolipoproteins B (ApoB) and the Phospholipid transfer protein (PLTP), providing new clues to the link between GALNT2 and lipid homeostasis. In contrast to the findings from the majority of previous studies, these results indicate that O-glycosylation can both decrease and increase substrate cleavage. We challenged this system with exogenous matrix metalloproteinase 9 (MMP9) and neutrophil elastase and observed substantial changes to the neutrophil elastase N-terminome, but no evidence for a similar role for MMP9. Overall, we show that loss of O-glycosylation leads to a general decrease in cleavage, that GalNAc-T2 O-glycosylation affects key regulators of the cellular proteolytic network, including multiple members of the Serpin family.