Presentation description

Proteins play a crucial role in nearly all biological processes, and understanding their structure is essential for uncovering their function and how they contribute to health and disease. Separation and identification of peptides is imperative for studying protein behavior and interactions. However, identifying and characterizing peptides is often complicated by the presence of isomeric forms. These subtle structural differences can significantly affect biological activity, yet they are difficult to distinguish using conventional mass spectrometry tools. Ion mobility spectrometry-mass spectrometry (IMS-MS) has been an increasingly popular technique used to resolve structurally similar molecules based on differences in size, charge, and shape. Recently, high-resolution IMS-MS-based techniques have demonstrated the separation of isotopomers and isotopologues based on changes in their mass distribution (i.e., changes in their center of mass and moments of inertia). This has enabled isomer-specific isotopic shifts by comparing the relative arrival times of heavy-labeled ions versus their light, unlabeled, counterparts. In this project, we use reductive amination to isotopically dimethylate various peptide isomers. This reaction will impart a heavy label at the N-terminus as well as every lysine, which we hypothesized would be enough to create unique mass distributions. To test if the isotopic shifts from dimethylated peptides would be isomer specific in nature, we constructed a simple eight sequence peptide isomer set which includes a lysine at varying positions. From our high-resolution IMS-MS separations of these peptide isotopologues, we observed isomer-specific mass distribution-based isotopic shifts as well as an inverse shift, where the heavier isotopologue arrived before the lighter one, when lysine was adjacent to the N-terminus. Overall, we have demonstrated how isotopic shifts can be an added tool for peptide isomer characterization and complements existing MS-based approaches.