Protein glycosylation analysis by HILIC-LC-MS of Proteinase K-generated N- and O-glycopeptides

Authors

  • Gerhild Zauner,

    1. Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands
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  • Carolien A. M. Koeleman,

    1. Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands
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  • André M. Deelder,

    1. Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands
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  • Manfred Wuhrer

    Corresponding author
    1. Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands
    • Leiden University Medical Center, Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center for Infectious Diseases, Postbus 9600, 2300 RC Leiden, The Netherlands Fax: +31-71-5266907
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Abstract

Analysis of protein glycosylation is essential in order to correlate certain disease types with oligosaccharide structures on proteins. Here, a method for the MS characterization of site-specific protein glycosylation is presented. Using asialofetuin and fetuin as model substances, a protocol for glycopeptide dissection was developed based on unspecific proteolysis by Proteinase K. The resulting glycopeptides were then resolved by nanoscale hydrophilic interaction liquid chromatography-electrospray multistage MS. The early elution range of O-glycopeptides was clearly separated from the late elution range of N-glycopeptides. Glycopeptides were analyzed by ion trap-MS/MS, which revealed fragmentations of glycosidic linkages and some peptide backbone cleavages; MS3 spectra predominantly exhibited cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N- and O-glycan attachment sites of fetuin could be confirmed; moreover using our method, the occupation of a new, additional O-glycosylation site serine 296 was found. In conclusion, this approach appears to be a valuable technique for in-depth analysis of the site-specific N-glycosylation and O-glycosylation of individual glycoproteins.

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