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Glycan size and attachment site location affect electron transfer dissociation (ETD) fragmentation and automated glycopeptide identification

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Abstract

We established a small synthetic N-glycopeptide library to systematically evaluate the effect of glycosylation site location and glycan size on the efficiency of electron transfer dissociation (ETD) fragmentation and subsequent automated identification. The glycopeptides within this library differed in glycosylation site position and glycan size ranging from the pentasaccharide N-glycan core to fully sialylated, biantennary N-glycans. Factors such as glycan size, glycosylation site position within a glycopeptide and individual precursor m/z all significantly impacted the number and quality of assignable glycopeptide backbone fragments. Generally, high charge/low m/z precursors (>3+) and glycopeptides carrying neutral, smaller N-glycans gave better product ion spectra, while hardly any product ions were detectable for sialylated, triply charged N-glycopeptides. These factors impacted correct glycopeptide identification by proteomics software tools such as SEQUEST or Amanda. A better understanding how glycopeptide physico-chemical properties influence fragmentation will help optimizing fragmentation conditions and generate better data, which will facilitate software assisted glycopeptide data analyses.

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Acknowledgements

We thank the Beilstein-Institut for supporting KA with a PhD scholarship and the Max Planck Society for financial support. DK is the recipient of an Australian Research Council Future Fellowship (project number FT160100344) funded by the Australian Government.

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Authors and Affiliations

  1. Institute for Glycomics, Griffith University, Building G26, Gold Coast Campus, Southport, QLD, 4222, Australia

    Kathirvel Alagesan & Daniel Kolarich

  2. Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14476, Potsdam, Germany

    Kathirvel Alagesan, Hannes Hinneburg, Peter H. Seeberger, Daniel Varón Silva & Daniel Kolarich

  3. Institute of Chemistry and Biochemistry, Freie Universität Berlin, 14195, Berlin, Germany

    Kathirvel Alagesan, Hannes Hinneburg & Peter H. Seeberger

  4. Department of Molecular Science, Faculty of Science and Engineering, Macquarie University, North Ryde, NSW, 2109, Australia

    Hannes Hinneburg

  5. ARC Centre for Nanoscale BioPhotonics, Adelaide, Australia

    Daniel Kolarich

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  1. Kathirvel Alagesan
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  2. Hannes Hinneburg
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  3. Peter H. Seeberger
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  4. Daniel Varón Silva
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  5. Daniel Kolarich
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Correspondence to Daniel Kolarich.

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Alagesan, K., Hinneburg, H., Seeberger, P.H. et al. Glycan size and attachment site location affect electron transfer dissociation (ETD) fragmentation and automated glycopeptide identification. Glycoconj J 36, 487–493 (2019). https://doi.org/10.1007/s10719-019-09888-w

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  • DOI: https://doi.org/10.1007/s10719-019-09888-w

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