Ion mobility-mass spectrometry (IM-MS) has emerged as a tool to characterize protein structures by measuring their m/z and cross-section. Despite the gas phase environment of ion mobility measurements, it is known that ions can retain native-like structures in the gas phase under ‘soft’ experimental settings. However, it remains unclear how long the native-like structure is retained in the gas phase.<br/>In this work, we employ our newly developed tandem-trapped ion mobility spectrometer-mass spectrometer (tTIMS-MS) to determine the unfolding kinetics of the protein ubiquitin in the gas phase. tTIMS-MS allows for ions to be stored in each of the mobility analyzers for a predetermined amount of time. In addition, tTIMS-MS can perform mobility selection prior to trapping, which allows for the unfolding kinetics of different subpopulations to be determined.<br/>We have successfully trapped ubiquitin in the first and second TIMS analyzer for up to 12s. Preliminary data show that folded, compact conformations can be retained for up to 12s in each of the TIMS analyzers for charge state 7+. By selecting different subpopulations of compact ubiquitin charge state 7+, we are able to show different unfolding kinetics for different subpopulations. These changes in the unfolding timescale compliment the idea that distinct conformations are present inside a single cross-section feature. Finally, we discuss how mobility selection prior to trapping experiments can be used to elucidate structural differences between different subpopulations.

Determining gas phase unfolding kinetics using tandem-trapped ion mobility spectrometry–mass spectrometry

ACS Spring Meeting 2021

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