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

## TL;DR

This study explains why some peptides have two distinct collisional cross-sectional area modes using machine learning and simulations.

## Contribution

The paper identifies basic site positioning as a key factor in determining peptide CCS modes.

## Key findings

- Peptides in the high CCS mode adopt extended, helical conformations.
- Low CCS mode peptides tend to have compact, globular structures.
- Protonation near the C-terminus supports helix formation in high CCS mode.

## Abstract

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.