Conformational Landscapes Observed through Controlled Kinetics (CLOCK): Precise Control of Ion Temperatures from Submillisecond to Second Time Scales.

A detailed understanding of ion conformational dynamics over a range of temperatures and time scales remains a central challenge in structural mass spectrometry. We recently introduced SLIMPHONY, which is a traveling-wave ion mobility (IM) instrument built on structures for lossless ion manipulations (SLIM), and demonstrated proof-of-concept experiments for conformational landscapes observed through controlled kinetics (CLOCK). Here, we develop CLOCK as a quantitative framework for probing ion dynamics on submillisecond to second time scales. We apply CLOCK to native-like ions of 6+ ubiquitin and 7+ cytochrome c and show that both the guard electrode potential and the duration of activation independently control the resulting conformational distributions. We then combine ion trajectory simulations with statistical methods to estimate effective translational temperatures, providing a physically interpretable scale for comparing activation conditions and a mechanistic understanding of CLOCK. Applying this temperature calibration to CLOCK experiments reveals that 6+ ubiquitin and 7+ cytochrome c exhibit very distinct unfolding kinetics over a similar range of effective temperatures. Together, these results establish CLOCK as a quantitative, temperature-resolved, and time-resolved framework for probing ion conformational dynamics.