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- Title
Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines.
- Authors
Steentoft, Catharina; Vakhrushev, Sergey Y; Vester-Christensen, Malene B; Schjoldager, Katrine T-B G; Kong, Yun; Bennett, Eric Paul; Mandel, Ulla; Wandall, Hans; Levery, Steven B; Clausen, Henrik
- 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 GalNAc? or NeuAc?2-6GalNAc? 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.
- Subjects
NUCLEASES; GLYCOSYLATION; ELECTROSPRAY ionization mass spectrometry; IMMUNOCYTOCHEMISTRY; GLYCOPROTEINS; CELL lines
- Publication
Nature Methods, 2011, Vol 8, Issue 11, p977
- ISSN
1548-7091
- Publication type
Article
- DOI
10.1038/nmeth.1731