Direct Characterization of Leucine and Isoleucine Residues in Peptides Using High-Resolution Tandem Mass Spectrometry

Leucine (Leu) and isoleucine (Ile), collectively termed Xle, can exert distinct biofunctional properties when present in the same position in a peptide sequence. Therefore, their accurate identification and assignment are critically important, especially to ensure the safety and efficiency of the peptide drugs. However, their identical molecular masses make it challenging to distinguish them by using mass spectrometry. Here, a high-resolution tandem mass spectrometric strategy based on a combination of high-energy collision dissociation (HCD) and collision-induced dissociation (CID) was developed to enable the direct and reliable assignment of Xle residues in peptides. Immonium ions (m/z 86.1) derived from Xle residues were first obtained using HCD, and then their fragmentation behavior was investigated in the m/z range 40–90 upon CID, in which two diagnostic product ions m/z 44.0 and m/z 69.0 were identified. Furthermore, their relative intensity (I₄₄/I₆₉) was newly found to be able to provide a robust basis for distinguishing Leu and Ile using an Orbitrap Eclipse Tribrid mass spectrometer. This strategy was successfully applied for the characterization of single and multiple Xle residues in various peptides, including peptides with different structures (linear and cyclic) and lengths (short and long). It can be readily applied to rapid de novo sequencing of peptides for drug research and development (R&D) and accurate identification of Leu/Ile isomeric impurities for drug quality control (QC).