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Analytical and Bioanalytical Chemistry

, Volume 407, Issue 22, pp 6637–6655 | Cite as

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

  • Martin Pattky
  • Simone Nicolardi
  • Beatrix Santiago-Schübel
  • Daniel Sydes
  • Yuri E. M. van der Burgt
  • Antonia N. Klein
  • Nan Jiang
  • Jeannine Mohrlüder
  • Karen Hänel
  • Janine Kutzsche
  • S. A. Funke
  • D. Willbold
  • S. Willbold
  • C. HuhnEmail author
Paper in Forefront

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.

Keywords

Extremely hydrophilic peptide Capillary electrophoresis Coating Electron transfer dissociation Alzheimer’s disease 

Notes

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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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Martin Pattky
    • 1
  • Simone Nicolardi
    • 2
  • Beatrix Santiago-Schübel
    • 1
  • Daniel Sydes
    • 1
  • Yuri E. M. van der Burgt
    • 2
  • Antonia N. Klein
    • 3
  • Nan Jiang
    • 3
  • Jeannine Mohrlüder
    • 3
  • Karen Hänel
    • 3
  • Janine Kutzsche
    • 3
  • S. A. Funke
    • 3
    • 4
  • D. Willbold
    • 3
    • 5
  • S. Willbold
    • 1
  • C. Huhn
    • 1
    • 6
    Email author
  1. 1.Central Institute for Engineering, Electronics, and Analytics (ZEA): Analytics (ZEA-3), Forschungszentrum Jülich GmbHJülichGermany
  2. 2.Leiden University Medical Center, Center for Proteomics and MetabolomicsLeidenThe Netherlands
  3. 3.Institute of Complex Systems, Structural Biochemistry (ICS-6)Research Centre JülichJülichGermany
  4. 4.BioanalytikHochschule für Angewandte WissenschaftenCoburgGermany
  5. 5.Institut für Physikalische BiologieHeinrich-Heine-Universität DüsseldorfDüsseldorfGermany
  6. 6.Institute for Physical and Theoretical ChemistryEberhard Karls Universität TübingenTübingenGermany

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