title: Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines.
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.
authors: Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors: Catharina
authors: C
authors: Steentoft
authors: Sergey Y
authors: SY
authors: Vakhrushev
authors: Malene B
authors: MB
authors: Vester-Christensen
authors: Katrine T-B G
authors: KT
authors: Schjoldager
authors: Yun
authors: Y
authors: Kong
authors: Eric Paul
authors: EP
authors: Bennett
authors: Ulla
authors: U
authors: Mandel
authors: Hans
authors: H
authors: Wandall
authors: Steven B
authors: SB
authors: Levery
authors: Henrik
authors: H
authors: Clausen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002241:Carbohydrates / Q000737:chemistry*; D002460:Cell Line; D002853:Chromatography, Liquid; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D020543:Proteome; D012689:Sequence Homology, Nucleic Acid; D053719:Tandem Mass Spectrometry
chemical_list: D002241:Carbohydrates; D020543:Proteome