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Glycan Side Reaction May Compromise ETD-Based Glycopeptide Identification

  • Research Article
  • Published:
Journal of The American Society for Mass Spectrometry

Abstract

Tris(hydroxymethyl)aminomethane (Tris) is one of the most frequently used buffer ingredients. Among other things, it is recommended and is usually used for lectin-based affinity enrichment of glycopeptides. Here we report that sialic acid, a common ‘capping’ unit in both N- and O-linked glycans may react with this chemical, and this side reaction may compromise glycopeptide identification when ETD spectra are the only MS/MS data used in the database search. We show that the modification may alter N- as well as O-linked glycans, the Tris-derivative is still prone to fragmentation both in ‘beam-type’ CID (HCD) and ETD experiments, at the same time—since the acidic carboxyl group was ‘neutralized’—it will display a different retention time than its unmodified counterpart. We also suggest solutions that—when incorporated into existing search engines—may significantly improve the reliability of glycopeptide assignments.

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Acknowledgments

The authors gratefully acknowledge the contribution of Ralf Schoepfer and Jonathan Trinidad in the mouse synaptosome study, and thank Marshall Bern for performing the Byonic database search, Adam Kerenyi for writing the script used for HCD peak list filtering, and Zoltan Kupihar for useful discussions and technical assistance.

K.F.M. was supported by NIH grant NIGMS 8P41GM103481, and by the Howard Hughes Medical Institute (to the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF, Director: A.L. Burlingame), and by the following grants: OTKA 105611 (to Z.D.), and BAROSS-DA07-DA-ESZK-07-2008-0036 (to the Biological Research Centre, HAS, director: P. Ormos). Z.D. was supported by the Janos Bolyai Fellowship of the HAS.

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Authors and Affiliations

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

    Zsuzsanna Darula & Katalin F. Medzihradszky

  2. Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA

    Katalin F. Medzihradszky

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  1. Zsuzsanna Darula
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  2. Katalin F. Medzihradszky
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Correspondence to Zsuzsanna Darula.

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Darula, Z., Medzihradszky, K.F. Glycan Side Reaction May Compromise ETD-Based Glycopeptide Identification. J. Am. Soc. Mass Spectrom. 25, 977–987 (2014). https://doi.org/10.1007/s13361-014-0852-9

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  • DOI: https://doi.org/10.1007/s13361-014-0852-9

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