Large Data Set Analysis Reveals Structural Origin of Peptide Collisional Cross Section Bimodal Behavior

Recent advances in ion mobility spectrometry have enabled the measurement of rotationally averaged collisional cross-sectional area (CCS) for millions of peptides as part of routine proteomic mass spectrometry workflows. One of the most striking findings in recent large ion mobility data sets is that CCS exhibits two distinct modes, most notably for charge 3⁺ peptides, with peptides predominantly exhibiting CCS in either the high or low mode. Here, using classical machine learning approaches, we identify that basic site positioning is a key sequence feature determining a peptide's CCS mode. Molecular dynamics simulations suggest that peptides in the high CCS mode tend to adopt more extended conformations and form charge-stabilized helical structures, whereas those in the low CCS mode adopt more compact, globular conformations. Further supporting this structural hypothesis, we provide evidence for preferential protonation near the C-terminus and uncover multiple position-dependent sequence determinants that all suggest the predominance of helix formation in the high CCS mode. Together, these findings will enable better integration of CCS measurements into protein identification and quantification pipelines, improving the performance of ion mobility-based proteomics.