Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines
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Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines. / Steentoft, Catharina; Vakhrushev, Sergey Y; Vester-Christensen, Malene B; Schjoldager, Katrine Ter-Borch Gram; Kong, Yun; Bennett, Eric Paul; Mandel, Ulla; Wandall, Hans; Levery, Steven B; Clausen, Henrik.
I: Nature Methods, Bind 8, Nr. 11, 10.2011, s. 977-82.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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 - 2011/10
Y1 - 2011/10
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 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.
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 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.
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
VL - 8
SP - 977
EP - 982
JO - Nature Methods
JF - Nature Methods
SN - 1548-7091
IS - 11
ER -
ID: 37622207