Optimization of High Field Asymmetric Waveform Ion Mobility Spectrometry to enhance the comprehensiveness of mass spectrometry-based proteomic analyses

The high complexity of biological samples can confound protein identification and compromise the depth and coverage of mass spectrometry-based proteomic analyses. Separation techniques that provide improved peak capacity and selectivity of LC-MS/MS analyses are often sought to overcome these limitations. High-field asymmetric waveform ion mobility spectrometry (FAIMS), a differential ion mobility device, has shown significant advantages by enhancing the transmission of multiple-charged peptide ions by reducing singly-charged interferences. In this context, the goal of this thesis was to explore the analytical capabilities of FAIMS to extend both the dynamic range of proteins/peptides detection and the precision of quantitative proteomic measurements by mass spectrometry. For this, we systematically integrated FAIMS in standard workflows to monitor the dynamic changes of the human proteome in response to hyperthermia. We first studied the merits of FAIMS to aid isobaric labeling quantification with tandem mass tags (TMT). This approach allows the labeling of peptide ions with different chemical groups of identical nominal masses but differing in their respective distribution of stable isotopes. TMT-labeled peptide ions produce reporter ions of distinct masses once fragmented by MS/MS. Unfortunately, the co-selection of precursor ions often leads to chimeric MS/MS spectra, and a slower MS3 centric approach is needed for precise quantification. Since FAIMS improves peak capacity by selectively transmitting ions based on their compensation voltage (CV), we obtained less peptide co-selection. FAIMS improved TMT quantification at the MS2 level and achieved 68 % more quantified peptides compared to regular LC-MS/MS, providing a deeper insight into the dynamic changes of the human proteome in response to heat stress. Further, we investigated stable isotope labeling by amino acids in cell culture (SILAC) quantification. If interferences co-elute simultaneously with SILAC isotopomers, quantification becomes inaccurate and SILAC counterparts can be missassigned to interfering ions in the highly populated chromatogram, thus skewing the SILAC ratio. FAIMS post-ionization fractionation could filter out background ions that can otherwise be attributed to a SILAC pair/triplet for quantification. In this work, FAIMS was especially beneficial for low abundant species and outperformed the standard strong cation exchange (SCX) fractionation workflow. In addition, FAIMS allowed the separation of phosphoisomers that are frequently observed in complex phosphoproteome extracts. The third aim of this work explored the charge state separation and enhanced transmission of highly charged peptides with FAIMS and its application for SUMOylated peptide analysis. FAIMS could enhance the transmission of triply charged SUMOylated peptides over typical tryptic peptide that are predominantly doubly charged, by applying more negative CVs with FAIMS. This allowed for gas-phase enrichment of SUMOylated peptide ions. FAIMS is an alternate and more straightforward approach to fractionate SUMOylated peptides that reduced sample loss, avoided sample processing, while increasing peak capacity in a more automated manner and added one order of magnitude in sensitivity. The last aim of this thesis was to improve the FAIMS instrumentation by interfacing it to the latest state-of-the-art instruments. With a new FAIMS device developed by our collaborators at Thermo Fisher Scientific, we demonstrate the robustness and the improved ion transmission for the new interface. In simple shotgun proteomics, FAIMS extended the dynamic range by one order of magnitude for deeper proteome coverage compared to regular LC-MS/MS. Moreover, fewer MS2 chimeric scans were generated with FAIMS gas-phase fractionation, which garnered more identifications and better quantification. For this, we directly compared LC-FAIMS-MS/MS to LC-MS/MS/MS using synchronous precursor selection (SPS) with and without basic reverse phase fractionation. Comparable quantitative measurements were obtained for all methods, except that FAIMS provided a 2.5-fold increase in the number of quantifiable peptides compared with non-FAIMS experiments. Overall, this thesis highlights some of the advantages that FAIMS can provide for proteomics experiments by improving both peptide identification and quantification.

Keyphrases: FAIMS, differential ion mobility, proteomics, mass spectrometry, TMT, SILAC, heat shock, phosphorylation, SUMOylation