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Nat Methods. 2011 Oct 9;8(11):977-82. doi: 10.1038/nmeth.1731.

Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines.

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1
Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

Abstract

Zinc-finger nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed analysis of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN targeting to truncate the O-glycan elongation pathway in human cells, generating stable 'SimpleCell' lines with homogenous O-glycosylation. Three SimpleCell lines expressing only truncated GalNAcα or NeuAcα2-6GalNAcα O-glycans were produced, allowing straightforward isolation and sequencing of GalNAc O-glycopeptides from total cell lysates using lectin chromatography and nanoflow liquid chromatography-mass spectrometry (nLC-MS/MS) with electron transfer dissociation fragmentation. We identified >100 O-glycoproteins with >350 O-glycan sites (the great majority previously unidentified), including a GalNAc O-glycan linkage to a tyrosine residue. The SimpleCell method should facilitate analyses of important functions of protein glycosylation. The strategy is also applicable to other O-glycoproteomes.

PMID:
21983924
DOI:
10.1038/nmeth.1731
[Indexed for MEDLINE]

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