Improved Methods for Recording Accurate Collision Cross Sections Using Cyclic Ion Mobility-Mass Spectrometry

Traveling wave ion mobility (TWIM) is a gas-phase separation technique widely used in structural biology to rapidly probe protein conformational states and in complex mixture analysis to catalyze an array of chemical measurements. Analyte size information can be extracted from TWIM data as collision cross section (CCS) values, which are physicochemical descriptors of the analytes. However, calibration using ions of known CCS is currently required. Recently, an improved TWIM calibration approach was introduced that incorporates blend and radial expressions derived from velocity relaxation and axial confinement effects. This approach offers several benefits over conventional power-law calibrations, including accurate calibration over a wide range of separation conditions and analyte classes using a universal calibrant set. While the blend and radial calibrations were extensively validated using a linear TWIM separator, next-generation cyclic ion mobility (cIM) offers additional benefits, such as scalable resolution using multipass separations and the ability to subject ions to multiple stages of activation and separation (i.e., IMSⁿ). The original work provided a brief application of the blend and radial expressions to cIM calibrations, but these cIM CCS calibrations were applied only to a basic single-pass mode of operation under limited separation conditions. Here, we describe a comprehensive method for accurate cIM calibrations using blend and radial expressions. Correcting ion arrival times for the time spent outside the separator and using conditions that minimize velocity relaxation effects reduced CCS measurement error. We also provide an optimized workflow for calibration using the blend and radial expressions in multipass and IMSⁿ modes of operation.