ExD identification of C-terminal Ile/Leu in natural peptides using d-ions.

Although mass spectrometry remains the most reliable and widely used method for peptide and protein sequencing in modern proteomics, important challenges remain. Among the most persistent is the differentiation between the isomeric leucine and isoleucine residues. Electron-transfer/higher-energy collision (EThcD) implemented on advanced Orbitrap platforms has demonstrated strong performance through detection of diagnostic w-ions, allowing one to distinguish leucine from isoleucine. However, the EThcD approach is less efficient for distinguishing these several amino acid residues at C-terminal positions. In this study, we test the hypothesis that d-ions, formed from primary a-ions, can be used to differentiate C-terminal leucine and isoleucine. Because d-ions are not prominent in EThcD, a combined electron-ion reaction-based dissociation (ExD) and ExD with additional collisional activation (ExciD) tandem mass spectrometry approach was employed and proved highly efficient. All 16 C-terminal Leu/Ile pairs were confidently identified in the sequences of nine natural peptides of the temporin family, isolated from skin secretions of the European common frog (Rana temporaria), based on detection of the corresponding d-ions. The remaining Leu/Ile residues in these peptides were differentiated via their characteristic w-ions, enabling complete sequence determination. The study showed that to minimize radical site migration, the supplemental collision energy should be kept as low as possible or increased stepwise to just below the migration threshold. In addition, a previously reported sequence of temporin M was corrected.