High-resolution tandem mass spectrometry strategy directly distinguishes Leucine and Isoleucine residues in peptides

Accurate identification and assignment of Leucine (Leu) and Isoleucine (Ile) residues, collectively termed Xle, are critically important for peptide drug safety and efficiency. Despite their identical molecular masses, these isomers can exert distinct biofunctional properties depending on their position within a peptide sequence. Traditional mass spectrometry struggles to differentiate them due to this mass identity, creating a significant analytical challenge in peptide drug research and development (R&D) and quality control (QC), where precise structural characterization is paramount.

Researchers developed a high-resolution tandem mass spectrometric strategy combining high-energy collision dissociation (HCD) and collision-induced dissociation (CID) to differentiate Leu and Ile. The method first used HCD to generate immonium ions (m/z 86.1) from Xle residues. Subsequently, the fragmentation behavior of these immonium ions was investigated using CID in the m/z range of 40-90. This analysis was performed on an Orbitrap Eclipse Tribrid mass spectrometer and applied to various peptides, including those with different structures (linear and cyclic) and lengths (short and long), to characterize single and multiple Xle residues.

The study successfully identified two diagnostic product ions, m/z 44.0 and m/z 69.0, resulting from the CID fragmentation of the m/z 86.1 Xle immonium ion. A key finding was that the relative intensity ratio (I44/I69) of these two diagnostic ions provided a robust basis for distinguishing between Leu and Ile. This novel approach was validated across diverse peptide samples. > The relative intensity ratio I44/I69 was newly identified as a robust basis for distinguishing Leu and Ile, successfully applied to linear and cyclic peptides of varying lengths. The strategy proved effective for characterizing both single and multiple Xle residues, demonstrating its broad applicability for accurate isomeric assignment.