Ion Mobility Separation of Isomeric Acyl-lysine Marks in Peptides

Isomeric post-translational modifications (PTMs) on proteins challenge proteomic analyses due to their identical mass and fragmentation patterns. We evaluate high-resolution ion mobility spectrometry (IMS) for separating three naturally occurring acyl-lysine isomer pairs on histones: crotonyl/methacryl, butyryl/isobutyryl, and l -/ d -lactyl. These PTMs were chemically installed on lysine residues 9 and 18 (K9 and K18) of synthetic histone H3 peptides (residues 3–15 and 3–25). Using trapped IMS (TIMS), we observe half-height separation of H3 [3–15] peptides possessing crotonyl/methacryl and l / d -lactyl marks, and the lactyl isomers of H3 [3–25] can be distinguished. In contrast, multipass cyclic IMS (cIM) achieves baseline or near-baseline resolution for every pair, except the longest butyryl/isobutyryl peptide isomers, despite their collision cross-section differences of ≈1%. We show that the resolution increased with the square root of the cIM pass number, allowing baseline separation within 300 ms. Beyond separation, structure-mobility relationships emerge: branched modifications (isobutyryl, methacryl) yield more compact gas-phase conformations than their linear analogs (butyryl, crotonyl). For the doubly crotonylated/methacrylated peptides studied, both PTM identity and site determine the mobility. These results demonstrate IMS as a sensitive method for the elucidation of the acyl-modified histone peptide fine structure by resolving isomeric PTM ambiguity. This addresses a persistent analytical bottleneck and should be included in routine proteomics MS-based workflows.