Two-dimensional gas-phase separations coupled to mass spectrometry for analysis of complex mixtures

Abstract

Ion mobility spectrometry (IMS) has been explored for decades, and its versatility in separation and identification of gas-phase ions is well established. Recently, field asymmetric waveform IMS (FAIMS) has been gaining acceptance in similar applications. Coupled to mass spectrometry (MS), both IMS and FAIMS have shown the potential for broad utility in proteomics and other biological analyses. A major attraction of these separations is extremely high speed, exceeding that of condensed-phase alternatives by orders of magnitude. However, modest separation peak capacities have limited the utility of FAIMS and IMS for analyses of complex mixtures. We report 2-D gas-phase separations that join FAIMS to IMS, in conjunction with high-resolution and accuracy time-of-flight (TOF) MS. Implementation of FAIMS/IMS and IMS/MS interfaces using electrodynamic ion funnels greatly improves sensitivity. Evaluation of FAIMS/IMS/TOF performance for a protein mixture tryptic digest reveals high orthogonality between FAIMS and IMS dimensions and, hence, the benefit of FAIMS filtering prior to IMS/MS. The effective peak capacities in analyses of tryptic peptides are approximately 500 for FAIMS/IMS separations and approximately 10(6) for 3-D FAIMS/IMS/MS, providing a potential platform for ultrahigh-throughput analyses of complex mixtures.

Fig. 1

Schematic of the ESI/FAIMS/IMS/Q-TOF MS instrumentation.

Fig. 2

Diagram of the FAIMS/IMS/MS experimental sequence.

Fig. 3

FAIMS CV spectrum for the test analyte mixture (central panel), and IMS/MS maps at 12 (out of 16 measured) CV values across the spectrum, as labeled (panels 1 – 12). All maps are for IMS drift times of 50 – 100 ms (horizontal axes) and m/z of 400 – 1200 (vertical axes). The four maps not shown were acquired at CV = 12.2, 12.5, 17.5 and 20.8 V.

Fig. 4

Summary IMS/MS map of test analyte compiled from CV-selected maps.

Fig. 5

Dispersion of distinct peptide identified in 3-D FAIMS/IMS/MS space.

Fig. 6

Distinct species (deisotoped data) found in selected IMS/MS maps of group I (circles, empty for CV = 7.5 V and filled for 9 V) and group II (triangles, empty for CV = 14 V and filled for 17 V).

Fig. 7

Dispersions of distinct peptide spots in the FAIMS/MS (a) and FAIMS/IMS (b) planes confirm the orthogonality of FAIMS separations to MS and IMS.