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

TY - JOUR

T1 - Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines

AU - Steentoft, Catharina

AU - Vakhrushev, Sergey Y

AU - Vester-Christensen, Malene B

AU - Schjoldager, Katrine Ter-Borch Gram

AU - Kong, Yun

AU - Bennett, Eric Paul

AU - Mandel, Ulla

AU - Wandall, Hans

AU - Levery, Steven B

AU - Clausen, Henrik

N1 - Erratum: Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines (Nature Methods (2011) 8 (977-982))

PY - 2011/11

Y1 - 2011/11

N2 - 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 GalNAcÎ ± or NeuAcÎ ±2-6GalNAcÎ ± 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.

AB - 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 GalNAcÎ ± or NeuAcÎ ±2-6GalNAcÎ ± 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.

KW - Amino Acid Sequence

KW - Base Sequence

KW - Carbohydrates

KW - Cell Line

KW - Chromatography, Liquid

KW - Glycosylation

KW - Humans

KW - Molecular Sequence Data

KW - Proteome

KW - Sequence Homology, Nucleic Acid

KW - Tandem Mass Spectrometry

UR - https://doi.org/10.1038/nmeth0215-160d

U2 - 10.1038/nmeth.1731

DO - 10.1038/nmeth.1731

M3 - Journal article

C2 - 21983924

SN - 1548-7091

VL - 8

SP - 977

EP - 982

JO - Nature Methods

JF - Nature Methods

IS - 11

ER -