A new strategy to separate peptide methionine sulfoxides stereoisomers for potential immunotherapy application

Peptide-based immunotherapy is a promising cancer treatment due to its scalability and patient-centered approach; therefore, there is an increasing focus on discovering neoantigens or modified peptides which could elicit a better immune response. We recently found that the methionine sulfoxide variant of YMDGTMSQV, an immunogenic tyrosinase derived epitope, elicits a stronger immune response compared to the native one. Here, we address the separation of six MHC I-restricted tyrosinase-derived peptides methionine sulfoxide stereoisomers (YMNGTMSQV, YMDGTMSQV, YMQGTMSQV, YMDGVMSQV, FMNGTMSQV, FMDGTMSQV) using offline two-dimensional high-performance liquid chromatography coupled with UV/Vis-Circular Dichroism detection. For all analyzed peptides, using our methodology, we observed that placing the sulfoxide on the methionine in sixth position results in no enantiodiscrimination, suggesting the net contribution of the N-terminus tyrosine or phenylalanine π electrons in separation. We show how modifying the amino acids in the vicinities of the methionine-sulfoxide residues results in the ablation of the chiral discrimination. We also render our methodology analytical to semi-preparative level. We describe the stereoisomers stability and capture differences regarding their propensity towards oxidation, our results suggesting that the substitution of the N-terminus tyrosine to phenylalanine could be involved in this process. We analyzed the tandem mass spectrometry fragmentation patterns of the separated optic isomers and search for clues about their discrimination. Moreover, we found that the stereoisomers are similar recognized by specific HLA compared with the racemic variant. Our methodology could be valuable for potential applications in an enantiomer-specific peptide-based immunotherapy selection.