FAIMS-IMS-QTOF MS Combined with TSPSO Deconvolution Algorithm for Effectively Probing Protein Conformation Changes...

Carbonic anhydrase (CA), a ∼29 kDa large protein, was systematically analyzed by using an r-FAIMS-IMS-QTOF MS system to probe the conformational changes induced by dipole locking in FAIMS. By operating the r-FAIMS in different modes (i.e., different voltage polarities), one dipole-locked and two unlocked CA FAIMS peaks were discovered for some charge states of the CA ions. Further IMS analyses for various charge state CA ions from different FAIMS peaks showed that the drift times for some of the same charge-state CA ions from dipole-locked and unlocked FAIMS peaks were completely different, implying different CA conformers. For some of the same charge-state CA ions, essentially the same drift times from the dipole-locked and unlocked FAIMS peaks were observed. These CA ions were further subjected to ion fragmentation (MS/MS) analyses in QTOF-MS. The product ion spectra for these CA ions were shown to be highly different under the same MS/MS operation conditions. These experimental results have positively confirmed that the same charge-state CA ions from dipole-locked and unlocked FAIMS peaks indeed represent different conformers. To achieve accurate IMS spectral analyses, all IMS overlapping peaks were deconvoluted by using a high-accuracy deconvolution algorithm (two-step particle swarm optimization algorithm, TSPSO). The r-FAIMS-IMS-QTOF MS system, combined with the TSPSO deconvolution algorithm developed through this study, can be a powerful platform for the structural elucidation of large biomolecules.