Gas-Phase Discrimination of Histidine Epimers in Peptides by Silver-Cation-Assisted Energy-Resolved Mass Spectrometry

Epimerization control of histidine residues during solid-phase peptide synthesis is a persistent challenge in the pharmaceutical industry. In this work, we demonstrate that energy-resolved mass spectrometry (ERMS) enables the direct differentiation and relative quantification of histidine epimers in intact peptides through stereochemistry-dependent gas-phase fragmentation. Peptides were analyzed as silver-cationized species, allowing Ag⁺ coordination to modulate fragmentation pathways according to the configuration of the histidine Cα center. Breakdown curves were generated for two epimeric tripeptide pairs and six heptapeptide epimer sets containing histidine at various sequence positions. In all cases, distinct and reproducible breakdown profiles were observed for l- and d-histidine-containing peptides, despite identical m/z values and primary sequences. Notably, epimeric content as low as 5% was readily detected within peptide mixtures, and comparison with reference breakdown curves enabled a preliminary estimation of epimer proportions. The observed differences are attributed to stereochemistry-dependent Ag⁺ binding geometries that influence histidine-centered dissociation channels, in contrast to the reference fragmentation experiments performed on the corresponding protonated ions. These results establish Ag⁺-assisted ERMS as a sensitive and broadly applicable gas-phase approach for probing histidine stereochemistry, offering a practical alternative to chromatographic- or derivatization-based methods for epimer analysis.