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Structure characterization of unexpected covalent O-sulfonation and ion-pairing on an extremely hydrophilic peptide with CE-MS and FT-ICR-MS

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Abstract

In this study, we characterized unexpected side-products in a commercially synthesized peptide with the sequence RPRTRLHTHRNR. This so-called peptide D3 was selected by mirror phage display against low molecular weight amyloid-β-peptide (Aβ) associated with Alzheimer’s disease. Capillary electrophoresis (CE) was the method of choice for structure analysis because the extreme hydrophilicity of the peptide did not allow reversed-phase liquid chromatography (RPLC) and hydrophilic interaction stationary phases (HILIC). CE-MS analysis, applying a strongly acidic background electrolyte and different statically adsorbed capillary coatings, provided fast and efficient analysis and revealed that D3 unexpectedly showed strong ion-pairing with sulfuric acid. Moreover, covalent O-sulfonation at one or two threonine residues was identified as a result of a side reaction during peptide synthesis, and deamidation was found at either the asparagine residue or at the C-terminus. In total, more than 10 different species with different m/z values were observed. Tandem-MS analysis with collision induced dissociation (CID) using a CE-quadrupole-time-of-flight (QTOF) setup predominantly resulted in sulfate losses and did not yield any further characteristic fragment ions at high collision energies. Therefore, direct infusion Fourier transform ion cyclotron resonance (FT-ICR) MS was employed to identify the covalent modification and discriminate O-sulfonation from possible O-phosphorylation by using an accurate mass analysis. Electron transfer dissociation (ETD) was used for the identification of the threonine O-sulfation sites. In this work, it is shown that the combination of CE-MS and FT-ICR-MS with ETD fragmentation was essential for the full characterization of this extremely basic peptide with labile modifications.

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Acknowledgments

The authors thank the Helmholtz Initiative and Networking Fund as well as the German Excellence Initiative commissioned by the German Research Foundation (DFG) for financial support.

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Authors and Affiliations

  1. Central Institute for Engineering, Electronics, and Analytics (ZEA): Analytics (ZEA-3), Forschungszentrum Jülich GmbH, 52428, Jülich, Germany

    Martin Pattky, Beatrix Santiago-Schübel, Daniel Sydes, S. Willbold & C. Huhn

  2. Leiden University Medical Center, Center for Proteomics and Metabolomics, 2333 ZA, Leiden, The Netherlands

    Simone Nicolardi & Yuri E. M. van der Burgt

  3. Institute of Complex Systems, Structural Biochemistry (ICS-6), Research Centre Jülich, 52425, Jülich, Germany

    Antonia N. Klein, Nan Jiang, Jeannine Mohrlüder, Karen Hänel, Janine Kutzsche, S. A. Funke & D. Willbold

  4. Bioanalytik, Hochschule für Angewandte Wissenschaften, 96450, Coburg, Germany

    S. A. Funke

  5. Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40225, Düsseldorf, Germany

    D. Willbold

  6. Institute for Physical and Theoretical Chemistry, Eberhard Karls Universität Tübingen, 72076, Tübingen, Germany

    C. Huhn

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  1. Martin Pattky
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Pattky, M., Nicolardi, S., Santiago-Schübel, B. et al. Structure characterization of unexpected covalent O-sulfonation and ion-pairing on an extremely hydrophilic peptide with CE-MS and FT-ICR-MS. Anal Bioanal Chem 407, 6637–6655 (2015). https://doi.org/10.1007/s00216-015-8826-8

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