Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines
Research output: Contribution to journal › Journal article › peer-review
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 GalNAca or NeuAca2-6GalNAca 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.
|Number of pages||6|
|Publication status||Published - Oct 2011|
Erratum: Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines (Nature Methods (2011) 8 (977-982))
- Amino Acid Sequence, Base Sequence, Carbohydrates, Cell Line, Chromatography, Liquid, Glycosylation, Humans, Molecular Sequence Data, Proteome, Sequence Homology, Nucleic Acid, Tandem Mass Spectrometry