Ion Mobility Separation of Isomeric Acyl-lysine Marks in Peptides

Abstract

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, 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, multi-pass 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 {approx}1%. We show that resolution increased with the square root of 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 elucidation of 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