Analyses of complex mixtures and characterization of ions increasingly involve gas-phase separations by ion mobility spectrometry (IMS) and particularly differential or field asymmetric waveform IMS (FAIMS) based on the difference of ion mobility in strong and weak electric fields. The key advantage of FAIMS is substantial orthogonality to mass spectrometry (MS), which makes FAIMS/MS hybrid a powerful analytical platform of broad utility. However, the potential of FAIMS has been constrained by limited resolution. Here, we report that the use of gas mixtures comprising up to 75% He dramatically increases the FAIMS separation capability, with the resolving power for peptides and peak capacity for protein digests reaching and exceeding 100. The resolution gains extend to small molecules, where previously unresolved isomers can now be separated. These performance levels open major new applications of FAIMS in proteomic and other biomolecular analyses.

Conventional ion mobility spectrometers that sample ion packets from continuous sources have traditionally been constrained by an inherently low duty cycle. As such, ion utilization efficiencies have been limited to <1% in order to maintain instrumental resolving power. Using a modified electrodynamic ion funnel, we demonstrated the ability to accumulate, store, and eject ions in conjunction with ion mobility spectrometry (IMS), which elevated the charge density of the ion packets ejected from the ion funnel trap (IFT) and provided a considerable increase in the overall ion utilization efficiency of the IMS instrument. A 7-fold increase in signal intensity was revealed by comparing continuous ion beam current with the amplitude of the pulsed ion current in IFT-IMS experiments using a Faraday plate. Additionally, we describe the IFT operating characteristics using a time-of-flight mass spectrometer attached to the IMS drift tube.

A new ion mobility spectrometer (IMS) platform was developed to improve upon the sensitivity and reproducibility of our previous platforms, and further enhance IMS-MS utility for broad ‘pan-omics’ measurements. The new platform incorporated an improved electrospray ionization source and interface for enhanced sensitivity, and providing the basis for further benefits based upon implementation of multiplexed IMS. The ion optics included electrodynamic ion funnels at both the entrance and exit of the IMS, an ion funnel trap for ion injection, and a design in which nearly all ion optics (e.g. drift rings, ion funnels) were fabricated using printed circuit board technology. The IMS resolving power achieved was ~73 for singly-charged ions, very close to the predicted diffusion-limited resolving power (~75). The platform’s performance evaluation (e.g. for proteomics measurements) include LC-IMS-TOF MS datasets for 30 technical replicates for a trypsin digested human serum, and included platform performance in each dimension (LC, IMS and MS) separately.

As the resolution of analytical methods improves, further progress tends to be increasingly limited by instrumental parameter instabilities that could be ignored before. This is now the case with differential ion mobility spectrometry (FAIMS), where fluctuations of the voltages and gas pressure have become critical. A new high-definition generator for FAIMS compensation voltage reported here provides a stable and accurate output than can be scanned with negligible steps. This reduces the spectral drift and peak width, thus improving the resolving power (R) and resolution. The gain for multiply-charged peptides that have narrowest peaks is up to ~40%, and R ~ 400 – 500 is achievable using He/N2 or H2/N2 gas mixtures.

A high-throughput approach and platform using 15 minute reversed-phase capillary liquid chromatography (RPLC) separations in conjunction with ion mobility spectrometry-mass spectrometry (IMS-MS) measurements was evaluated for the rapid analysis of complex proteomics samples. To test the separation quality of the short LC gradient, a sample was prepared by spiking twenty reference peptides at varying concentrations from 1 ng/mL to 10 µg/mL into a tryptic digest of mouse blood plasma and analyzed with both a LC-Linear Ion Trap Fourier Transform (FT) MS and LC-IMS-TOF MS. The LC-FT MS detected thirteen out of the twenty spiked peptides that had concentrations ≥100 ng/mL. In contrast, the drift time selected mass spectra from the LC-IMS-TOF MS analyses yielded identifications for nineteen of the twenty peptides with all spiking levels present. The greater dynamic range of the LC-IMS-TOF MS system could be attributed to two factors. First, the LC-IMS-TOF MS system enabled drift time separation of the low concentration spiked peptides from the high concentration mouse peptide matrix components, reducing signal interference and background, and allowing species to be resolved that would otherwise be obscured by other components. Second, the automatic gain control (AGC) in the linear ion trap of the hybrid FT MS instrument limits the number of ions that are accumulated to reduce space charge effects and achieve high measurement accuracy, but in turn limits the achievable dynamic range compared to the IMS-TOF instrument.