The inherent structural heterogeneity of biomolecules is an important biophysical property that is essential to their function, but is challenging to characterize experimentally. We present a workflow that rapidly and quantitatively assesses the conformational heterogeneity of peptides and proteins in the gas phase using traveling wave ion mobility (TWIM) arrival time distributions (ATDs). We have established a set of semi-empirical equations that model the TWIM ATD peak width and resolution across a wide range of wave amplitudes (V) and wave velocities (v). In addition, a conformational broadening parameter, δ, can be extracted from this analysis that reports on the contribution of conformational heterogeneity to the broadening of TWIM ATD peak width during ion mobility separation. We use this δ value to evaluate the conformational heterogeneity of a set of helical peptides, and our analysis correlates well with previous peak width observations reported for these ions. Furthermore, we use molecular dynamics simulations to independently investigate the general flexibility of these peptides in the gas phase, and generate similar trends found in experimental TWIM data. Finally, we extended our analysis to Avidin, a 64-kDa homotetramer, and quantify the structural heterogeneity of this intact complex using TWIM ATD data as a function of cross-linking. We observe an initial reduction in δ values as a function of cross-linker concentration, demonstrating the sensitivity of our δ value analysis to changes in flexibility of the assembly.
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