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A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation

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Hägglund, Per ; Bunkenborg, Jakob ; Elortza, Felix ; Jensen, Ole Nørregaard ; Roepstorff, Peter. / A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation. In: Journal of Proteome Research. 2004 ; Vol. 3, No. 3. pp. 556-66.

Bibtex

@article{ecbff19ce8cd4c1da88405faf9f8a8e1,
title = "A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation",
abstract = "Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-beta-N-acetylglucosaminidases (EC 3.2.1.96). After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by lectin chromatography followed by 1D gel-electrophoresis for determination of 62 glycosylation sites in 37 glycoproteins.",
keywords = "Acetylglucosamine, Amino Acid Sequence, Asparagine, Chromatography, Liquid, Glycosylation, Humans, Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase, Mass Spectrometry, Molecular Sequence Data, Plasma",
author = "Per H{\"a}gglund and Jakob Bunkenborg and Felix Elortza and Jensen, {Ole N{\o}rregaard} and Peter Roepstorff",
year = "2004",
language = "English",
volume = "3",
pages = "556--66",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",
number = "3",

}

RIS

TY - JOUR

T1 - A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation

AU - Hägglund, Per

AU - Bunkenborg, Jakob

AU - Elortza, Felix

AU - Jensen, Ole Nørregaard

AU - Roepstorff, Peter

PY - 2004

Y1 - 2004

N2 - Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-beta-N-acetylglucosaminidases (EC 3.2.1.96). After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by lectin chromatography followed by 1D gel-electrophoresis for determination of 62 glycosylation sites in 37 glycoproteins.

AB - Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-beta-N-acetylglucosaminidases (EC 3.2.1.96). After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by lectin chromatography followed by 1D gel-electrophoresis for determination of 62 glycosylation sites in 37 glycoproteins.

KW - Acetylglucosamine

KW - Amino Acid Sequence

KW - Asparagine

KW - Chromatography, Liquid

KW - Glycosylation

KW - Humans

KW - Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase

KW - Mass Spectrometry

KW - Molecular Sequence Data

KW - Plasma

M3 - Journal article

VL - 3

SP - 556

EP - 566

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 3

ER -

ID: 42362767