High-Resolution Ion Mobility-Based Isotopic Shifts of Dimethylated Peptide Isomers

Protein and peptide isomerization arising from sequence variation, epimerization, or post-translational modification directly influences biological structure and function, necessitating rapid and reliable methods for isomer delineation. Ion mobility spectrometry-mass spectrometry (IMS-MS) provides fast, gas-phase separations of peptide isomers, yet conventional mobility measurements offer limited information beyond bulk structural changes (i.e., compaction or elongation). Comparatively, isotopic shifts, or the measurement of differences in arrival time between light and heavy isotopologues, have been shown to be orthogonal to conventional IMS-MS separations as well as able to probe ion fine structure related to changes in center of mass and moments of inertia. In this work, we probed the use of isotopic dimethylation to introduce isomer-specific isotopic shifts as measured with high-resolution cyclic ion mobility separations. We observed that shifts in arrival time were diagnostic for a range of peptide isomers, including lysine positional isomers and β-amyloid aspartic acid isomers. The magnitude and direction of these shifts varied systematically with peptide sequence and, in several cases, defied predictions based solely on reduced mass theory. Molecular modeling revealed that these effects arise from isomer-dependent changes in mass distribution, specifically alterations in center of mass and moments of inertia after isotopic labeling. We also observed direct orthogonality of our isotopic shifts to absolute arrival times, highlighting how our strategy can provide an additional dimension of information to traditional IMS-MS measurements. Overall, isotopic dimethylation-based isotopic shifts offer a broadly applicable and structurally sensitive complement to the existing IMS-MS toolbox for peptide isomer characterization.