Improving Ion Mobility Mass Spectrometry of Proteins through Tristate Gating and Optimization of Multiplexing...

Coupling drift tube ion mobility (IM) to Fourier transform mass spectrometry (FT-MS) affords the opportunity for gas-phase separation of ions based on size and conformation with high-resolution mass analysis. However, combining IM and FT-MS is challenging because ions exit the drift tube on a much faster time scale than the rate of mass analysis. Fourier transform (FT) and Hadamard transform multiplexing methods have been implemented to overcome the duty-cycle mismatch, offering new avenues for obtaining high-resolution, high-mass-accuracy analysis of mobility-selected ions. The gating methods used to integrate the drift tube with the FT mass analyzer discriminate against the transmission of large, low-mobility ions owing to the well-known gate depletion effect. Tristate gating strategies have been shown to increase ion transmission for drift tube IM-FT-MS systems through implementation of dual ion gating, controlling the quantity and timing of ions through the drift tube to reduce losses of slow-moving ions. Here we present an optimized set of multiplexing parameters for tristate gating ion mobility of several proteins on an Orbitrap mass spectrometer and further report parameters for increased ion transmission and mobility resolution as well as decreased experimental times from 15 min down to 30 s. On average, peak intensities in the arrival time distributions (ATDs) for ubiquitin increased 2.1× on average, while those of myoglobin increased by 1.5× with a resolving power increase on average of 11%.