title: Structure characterization of unexpected covalent O-sulfonation and ion-pairing on an extremely hydrophilic peptide with CE-MS and FT-ICR-MS.
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.
authors.affiliation: Central Institute for Engineering, Electronics, and Analytics (ZEA): Analytics (ZEA-3), Forschungszentrum Jülich GmbH, 52428, Jülich, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Pattky
authors.forename: Simone
authors.initials: S
authors.lastname: Nicolardi
authors.forename: Beatrix
authors.initials: B
authors.lastname: Santiago-Schübel
authors.forename: Daniel
authors.initials: D
authors.lastname: Sydes
authors.forename: Yuri E M
authors.initials: YE
authors.lastname: van der Burgt
authors.forename: Antonia N
authors.initials: AN
authors.lastname: Klein
authors.forename: Nan
authors.initials: N
authors.lastname: Jiang
authors.forename: Jeannine
authors.initials: J
authors.lastname: Mohrlüder
authors.forename: Karen
authors.initials: K
authors.lastname: Hänel
authors.forename: Janine
authors.initials: J
authors.lastname: Kutzsche
authors.forename: S A
authors.initials: SA
authors.lastname: Funke
authors.forename: D
authors.initials: D
authors.lastname: Willbold
authors.forename: S
authors.initials: S
authors.lastname: Willbold
authors.forename: C
authors.initials: C
authors.lastname: Huhn
mesh_terms: D001665:Binding Sites; D019075:Electrophoresis, Capillary / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013431:Sulfates / Q000737:chemistry; D013451:Sulfonic Acids / Q000737:chemistry*
chemical_list: D010455:Peptides; D013431:Sulfates; D013451:Sulfonic Acids