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Using “spectral families” to assess the reproducibility of glycopeptide enrichment: human serum O-glycosylation revisited

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Abstract

Growing evidence on the diverse biological roles of extracellular glycosylation as well as the need for quality control of protein pharmaceuticals make glycopeptide analysis both exciting and important again after a long hiatus. High-throughput O-glycosylation studies have to tackle the complexity of glycosylation as well as technical difficulties and, up to now, have yielded only limited results mostly from single enrichment experiments. In this study, we address the technical reproducibility of the characterization of the most prevalent O-glycosylation (mucin-type core 1 structures) in human serum, using a two-step lectin affinity-based workflow. Our results are based on automated glycopeptide identifications from higher-energy C-trap dissociation and electron transfer dissociation MS/MS data. Assignments meeting strict acceptance criteria served as the foundation for generating “spectral families” incorporating low-scoring MS/MS identifications, supported by accurate mass measurements and expected chromatographic retention times. We show that this approach helped to evaluate the reproducibility of the glycopeptide enrichment more reliably and also contributed to the expansion of the glycoform repertoire of already identified glycosylated sequences. The roadblocks hindering more in-depth investigations and quantitative analyses will also be discussed.

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Acknowledgments

The authors are grateful to Jonathan Trinidad and Jason Maynard for their useful advice on the WGA immobilization. We would like to thank Agnes Arva for the technical assistance. This work was supported by the following grants: 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); and New Szechenyi Plan GOP-1.1.1-11-2012-0452.

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Authors and Affiliations

  1. Laboratory of Proteomics Research, Biological Research Centre, Hungarian Academy of Sciences, Temesvari krt 62, 6726, Szeged, Hungary

    Adam Pap, Katalin F. Medzihradszky & Zsuzsanna Darula

  2. Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, 600 16th Street, Genentech Hall N474A, San Francisco, CA, 94158-2517, USA

    Katalin F. Medzihradszky

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Correspondence to Zsuzsanna Darula.

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Published in the topical collection Glycomics, Glycoproteomics and Allied Topics with guest editors Yehia Mechref and David Muddiman.

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Pap, A., Medzihradszky, K.F. & Darula, Z. Using “spectral families” to assess the reproducibility of glycopeptide enrichment: human serum O-glycosylation revisited. Anal Bioanal Chem 409, 539–550 (2017). https://doi.org/10.1007/s00216-016-9960-7

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