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.