General Method to Obtain Collision Cross-Section Values in Multipass High-Resolution Cyclic Ion Mobility Separations

In recent years, ion mobility spectrometry-mass spectrometry (IMS-MS) has advanced the field of omics-based research, especially with the development of high-resolution platforms; however, these separations have generally been qualitative in nature. The rotationally averaged ion neutral collision cross section (CCS) is one of the only quantitative metrics available for aiding in characterizing biomolecules in IMS-MS. However, determining the CCS of an ion for multipass IMS systems, such as in cyclic ion mobility-mass spectrometry (cIMS-MS) and structures for lossless ion manipulations, has been challenging due to the lack of methods available for calculating CCS when more than a single pass is required for separation as well as the laborious nature of requiring calibrants and unknown compounds to be subjected to identical number of passes, which may not be possible in certain instances because of peak splitting, high levels of diffusion, etc. Herein, we present a general method that uses average ion velocities for calculating CCS values in cIMS-MS-based separations. Initially, we developed calibration curves using common CCS calibrants [i.e., tetra-alkylammonium salts, polyalanine, and hexakis(fluoroalkoxy)phosphazines] at different traveling wave (TW) conditions and the calculated cIMS CCS values were within ∼1% error or less compared to previously established drift tube IMS CCS measurements. Since it has been established that glycans can split into their α/β anomers, we utilized this method for two glycan species, 2α-mannobiose and melibiose. Both glycans were analyzed at the same TW conditions as the calibrants, and we observed anomer splitting at pathlengths of 20 m for 2α-mannobiose and 40 m for melibiose and thus assigned two unique CCS values for each glycan, which is the first time this has ever been done. We have demonstrated that the use of average ion velocities is a robust approach for obtaining CCS values with good agreement to CCS measurements from the previous literature and anticipate that this methodology can be applied to any IMS-MS platform that utilizes multipass separations. Our future work aims to incorporate this methodology for the development of a high-resolution CCS database to aid in the characterization of human milk oligosaccharides.