Tandem FAIMS and IMS Combined with MS/MS for Effectively Probing the Structural Diversity of Proteins.

A three-dimensional gas-phase ion separation instrument platform that integrates high-sensitivity and high-resolution racetrack field asymmetric waveform ion mobility spectrometry (r-FAIMS) with quadrupole time-of-flight mass spectrometry (Agilent 6560 IMS-QTOF MS) was developed to probe the conformational diversity of cytochrome C ions. A charge state envelope of +8 to +19 for the cytochrome C ions was observed in the acquired MS spectrum. For the +8 to +11 charge states of cytochrome C ions, multiple peaks were clearly observed in the IMS spectra, while only one peak was observed in the corresponding FAIMS spectra. In contrast, well-separated multiple peaks were clearly observed for the +13 to +19 charge states of cytochrome C ions in the FAIMS spectra. However, the ions from different FAIMS peaks were shown to have very close drift times in the second dimension IMS measurements, implying that these ions had essentially the same collisional cross-sectional areas. To verify these FAIMS peaks, corresponding to different compensation voltages, for the +13 to +19 charge states of cytochrome C ions indeed represent different protein conformers. The CV-selected cytochrome C ions were further subjected to ion fragmentation (MS/MS) analysis in the third dimension QTOF MS. MS/MS analyses have clearly demonstrated that the product ion spectra for the same charge state of cytochrome C ions from different FAIMS CV peaks are highly different under the same MS/MS operation conditions (i.e. under the same collision energy). The results from the MS/MS analyses thus convincingly prove that the protein ions from different FAIMS peaks are different conformers. Gas-phase ion separations in FAIMS and IMS are, thus, highly orthogonal. Unique to any other alternatives, the proposed r-FAIMS-IMS-MS/MS technique allows for a highly efficient FAIMS separation of protein structural isomers, detailed IMS measurements of the collisional cross-sectional area for each isomeric protein, and direct confirmation of isomeric proteins via MS/MS.