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O-glycosylation sites identified from mucin core-1 type glycopeptides from human serum

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Abstract

In this work O-linked glycopeptides bearing mucin core-1 type structures were enriched from human serum. Since about 70 % of the O-glycans in human serum bind to the plant lectin Jacalin, we tested a previously successful protocol that combined Jacalin affinity enrichment on the protein- and peptide-level with ERLIC chromatography as a further enrichment step in between, to eliminate the high background of unmodified peptides. In parallel, we developed a simpler and significantly faster new workflow that used two lectins sequentially: wheat germ agglutinin and then Jacalin. The first lectin provides general glycopeptide enrichment, while the second specifically enriches O-linked glycopeptides with Galβ1-3GalNAcα structures. Mass spectrometric analysis of enriched samples showed that the new sample preparation method is more selective and sensitive than the former. Altogether, 52 unique glycosylation sites in 20 proteins were identified in this study.

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Acknowledgments

The authors are grateful to Andrew Alpert for the generous gift of the PolyWAX column, Samuel Myers for the WGA-POROS column, ProteinMetrics Inc. for access to the Byonic software. We would like to thank Agnes Arva for technical assistance. We appreciate the help of Robert Chalkley in editing our manuscript. This work was supported by the following grants: National Institutes of Health NIGMS 8P41GM103481, Hungarian Scientific Research Fund 105611 (to Z. Darula), National Research, Development and Innovation Fund BAROSS-DA07-DA-ESZK-07-2008-0036 (to the Biological Research Centre, HAS, director: P. Ormos), New Szechenyi Plan GOP-1.1.1-11-2012-0452, and by the Howard Hughes Medical Institute (to the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF, Director: A. L. Burlingame). Z. Darula was supported by the Janos Bolyai Fellowship of the Hungarian Academy of Sciences.

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Authors and Affiliations

  1. Laboratory of Proteomics Research, Biological Research Centre of the Hungarian Academy of Sciences, Institute of Biochemistry, Szeged, Hungary

    Zsuzsanna Darula, Farkas Sarnyai & Katalin F. Medzihradszky

  2. Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, USA

    Katalin F. Medzihradszky

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  1. Zsuzsanna Darula
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  2. Farkas Sarnyai
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  3. Katalin F. Medzihradszky
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Correspondence to Zsuzsanna Darula.

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Darula, Z., Sarnyai, F. & Medzihradszky, K.F. O-glycosylation sites identified from mucin core-1 type glycopeptides from human serum. Glycoconj J 33, 435–445 (2016). https://doi.org/10.1007/s10719-015-9630-6

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