Using Cyclic Traveling Wave Ion Mobility Mass Spectrometry to Investigate native and Deuterated Proteins and peptides - new capabilities for core labs

A problem that many labs face is the long analysis time required by chromatography. The introduction of hybrid ion mobility-TOF mass spectrometers has proven advantageous in retaining peak capacity when chromatographic times are shortened, due to the extra dimension of separation afforded by the ion mobility (IM) device. We investigated the utility of cIM for peptide separation under fast chromatographic conditions and by direct infusion to determine if the chromatographic separation time could be reduced or eliminated, and still retain the peak capacity for intact proteins and digests. An enolase tryptic digest and peptide P1 (HHHHHHIIKIIK) were used as model systems in this study. Experiments were performed on a cyclic ion mobility-enabled quadrupole time-of-flight (Q-cIM-oa ToF) mass spectrometer. . The cIM device has a circular path that provides a longer, higher mobility resolution separation path (1m) whilst a multi-pass capability provides significantly higher path length and hence resolution over a reduced (selected) mobility range. Data were acquired in both infusion and LC-MS modes. The enolase digest was used as a model system for the separation of peptides by rapid LC-MS. 82 % sequence coverage was obtained with a gradient of less than 3 minutes, afforded by the enhanced mobility and TOF resolution. Partially and fully co-eluting species were separated by a single pass through the cIM device, even with this short chromatographic gradient, useful for data-independent LC-MS workflows. We used the model peptide P1 to also assess hydrogen/deuterium back exchange within the cIM device. The deuterium level in the peptide changed depending on the number of passes in the IM device: more passes resulted in decreased average deuterium. This work will demonstrate how ion mobility MS and multiple stages of IM can dramatically increase peak capacity without additional separation time while adding new structural information to separated proteins and peptides.