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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Carbohydrates / chemistry*
  • Cell Line
  • Chromatography, Liquid
  • Glycosylation
  • Humans
  • Molecular Sequence Data
  • Proteome*
  • Sequence Homology, Nucleic Acid
  • Tandem Mass Spectrometry


  • Carbohydrates
  • Proteome