In this thesis, a miniaturised field asymmetric waveform ion mobility spectrometry (FAIMS) device is combined with mass spectrometry (MS), and liquid chromatography, for the development and application of bioanalytical methodologies. FAIMS is a highly orthogonal to MS and LC and has the potential to enhance both targeted and non-targeted bioanalytical applications. Chapter two demonstrates the capability of the FAIMS combined with mass spectrometry to reduce the complexity of the mass spectrum by separating species of different charge states and overlapping mass-to-charge ratios that are challenging to separate by MS. FAIMS selected transmission shows improvement in signal-to-noise ratios for low intensity species and enables visualisation of species undetectable without FAIMS. Chapter three describes the development of an LC-FAIMS-MS method for the rapid analysis of saliva for the identification of potential biomarkers as a result of oxidative stress. The combination of FAIMS showed a reduction in saliva matrix interferences resulting in improved discrimination and peak integration of two salivary oxypurine compounds in a rapid LC-FAIMS-MS method. Chapter four investigates the FAIMS separation of seven steroid metabolites with a range of cationic adducts, in order to develop a rapid screening LC-FAIMS-MS method for the determination of isobaric steroid metabolites in urine. LC-FAIMS-MS analysis of the steroid metabolites shows improved discrimination of co-eluting and isobaric steroid metabolites with improvements in signal-to-noise ratio with reductions in chemical noise, demonstrating the potential of combining FAIMS with LC-MS. Chapter five demonstrates the potential of FAIMS to increase peak capacity in non-targeted omics applications, by combining rapid compensation field scanning of the FAIMS with ultra-high performance LC-MS. The rapid scanning of the FAIMS allows acquisition of full scan FAIMS and MS nested data sets within the timescale of a UHPLC chromatographic peak, and is applied to the non-targeted profiling of human urine. Improvements in the number of features detected using LC-FAIMS-MS were as a result of reductions in chemical noise and separation of co-eluting isobaric species across the whole analytical space, demonstrating the potential of combining FAIMS with LC and MS.

In this thesis, the performance of a miniaturised field asymmetric waveform ion mobility spectrometry (FAIMS) device hyphenated with time-of-flight mass spectrometry is studied and evaluated for analysis of a variety of compounds in different sample matrices. FAIMS is a selective spectrometer which is highly orthogonal to mass spectrometry and has the potential for enhancing sensitivity and improve selectivity of rapid analyses. In Chapter 2, the performance of the miniaturised FAIMS device is tested for stability and transmission under a wide range of ion source conditions. An investigation of three different systems, including pairs of isobaric, isomeric and near-mass ions shows that miniaturised FAIMS has the ability to distinguish between analytes that are challenging to separate by mass spectrometry. Chapter 3 explores the effect of changing the composition of the carrier gas by observing the effect of adding gas modifiers on the FAIMS spectra of small molecules, peptides and proteins. Chapter 4 investigates the advantages of combining a fast FAIMS separation with mass spectrometry in the analysis of nitrogen-containing pharmaceutical impurities, where FAIMS is found to offer additional selectivity. In Chapter 5, the development of a UHPLC-FAIMS-MS method for the quantitative determination of a drug metabolite in urine is reported. UHPLC-FAIMS-MS shows improvements in signal-to noise and linear dynamic range as well as a reduction in chemical noise, demonstrating the potential of combining FAIMS with mass spectrometry.

Field asymmetric waveform ion mobility spectrometry (FAIMS) is a gas phase atmospheric pressure separation technique that exploits the difference in the mobility of ions in alternating low and high electric fields as they are carried between two electrodes. In this thesis, a miniaturised FAIMS separation step has been applied to increase selectivity, enhance sensitivity and improve the quality of mass spectral data for rapid, high-throughput protein and peptide analysis. In Chapter 2, charge state separations were used to generate pseudo-peptide mass fingerprint data by FAIMS-MS, permitting confident protein identification using ESI sample introduction as an alternative to MALDI-TOF-MS methods. In addition, pre-cursor ions were targeted prior to MS/MS analysis. Chapter 3 describes the analysis of intact proteins by miniaturised FAIMS-MS. Multiple charge states of intact proteins were separated on the basis of differences in differential mobility. Higher charge states were found to be transmitted at similar CVs suggesting that the miniaturised FAIMS device was separating ions on the basis of 3D structure. In addition, multiple species could be observed at the same m/z suggesting the presence of different protein conformers. In Chapter 4, miniaturised FAIMS was used to select ions on the basis of differential mobility prior to in-source collision-induced dissociation CID, LC and ToF-MS analysis for qualitative and quantitative analysis of peptides mixtures. This was applied to the analysis of co-eluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching.

Indiana University-Purdue University Indianapolis (IUPUI) / Cachexia is a devastating muscle wasting syndrome affecting multiple biochemical pathways and is a comorbidity of many diseases including pancreatic ductal adenocarcinoma (PDAC). PDAC patients with cachexia commonly experience systemic inflammation, progressive loss of lean muscle and adipose tissue, and cardiac dysfunction. The present workflow identifies proteins and their post-translational modifications extracted from both cardiac and skeletal muscle tissue isolated from a murine model of PDAC-associated cachexia. Reported here are differentially occurring post-translational modifications found on the most abundant contractile proteins. Tissue from mouse muscle samples were collected two weeks after either receiving a sham surgery or orthotopically implanted with PDAC tumor cells, with or without a follow-up chemotherapy treatment of the standard of care agent gemcitabine with nab-Paclitaxel. Whole tissue blocks of gastrocnemius or heart were either flash frozen and pulverized or homogenized in denaturing lysis buffer and then sonicated to facilitate protein extraction. After disulfide bond reduction, cysteine alkylation, and trypsin digestion, the resultant peptides were subjected to molecular barcoding using tandem mass tag isobaric labeling reagents to facilitate multiplexing. The first and second dimension of peptide separation in the multiplexed sample is accomplished with an offline, high pH, reverse phase (RP)-LC fractionation followed by an online RP-LC at lower pH. The use of high-field asymmetric-waveform ion mobility spectrometry provided a last dimension of separation before MSn analyses. ​This novel, proteomic workflow enables deep proteome profiling in the progression of cancer-induced cachexia. The use of multi-dimensional chromatographic separation and differential ion mobility technique have allowed us to identify almost 4,500 proteins groups of gastrocnemius muscle tissue and nearly 7,100 protein groups of myocardium taken from the murine PDAC model of pancreatic cancer. A comprehensive analysis of the data collected from this workflow was used to calculate differential post-translational modifications on major contractile proteins isolated from PDAC model muscle tissue, with or without chemotherapy, when compared to sham surgery controls. Differential post-translational modifications and protein expression changes found to contribute to cancer cachexia may elucidate novel molecular mechanisms and cellular signaling that drive cachexia progression. / 2022-08-23

La phosphorylation est une modification post-traductionnelle modulant l’activité, la conformation ou la localisation d’une protéine et régulant divers processus. Les kinases et phosphatases sont responsables de la dynamique de phosphorylation et agissent de manière coordonnée. L’activation anormale ou la dérégulation de kinases peuvent conduire au développement de cancers ou de désordres métaboliques. Les récepteurs tyrosine kinase (RTKs) sont souvent impliqués dans des maladies et la compréhension des mécanismes régissant leur régulation permet de déterminer les effets anticipés sur leurs substrats. Dans ce contexte, le but de cette thèse est d’identifier les évènements de phosphorylation intervenant dans la voie de l’insuline chez la drosophile impliquant un RTK : le récepteur de l’insuline (InR). La cascade de phosphorylation déclenchée suite à l’activation du récepteur est conservée chez le mammifère. Afin d’étudier le phosphoprotéome de cellules S2 de drosophile, nous avons utilisé une étape d’enrichissement de phosphopeptides sur dioxyde de titane suivie de leur séparation par chromatographie liquide (LC) et mobilité ionique (FAIMS). Les phosphopeptides sont analysés par spectrométrie de masse en tandem à haute résolution. Nous avons d’abord démontré les bénéfices de l’utilisation du FAIMS comparativement à une étude conventionnelle en rapportant une augmentation de 50 % dans le nombre de phosphopeptides identifiés avec FAIMS. Cette technique permet de séparer des phosphoisomères difficilement distinguables par LC et l’acquisition de spectres MS/MS distincts où la localisation précise du phosphate est déterminée. Nous avons appliqué cette approche pour l’étude des phosphoprotéomes de cellules S2 contrôles ou traitées à l’insuline et avons identifié 32 phosphopeptides (sur 2 660 quantifiés) pour lesquels la phosphorylation est modulée. Étonnamment, 50 % des cibles régulées possèdent un site consensus pour la kinase CK2. Une stratégie d’inhibition par RNAi a été implémentée afin d’investiguer le rôle de CK2 dans la voie de l’insuline. Nous avons identifié 6 phosphoprotéines (CG30085, su(var)205, scny, protein CDV3 homolog, D1 et mu2) positivement régulées suite à l’insuline et négativement modulées après le traitement par RNAi CK2. Par essai kinase in vitro, nous avons identifié 29 cibles directes de CK2 dont 15 corrélaient avec les résultats obtenus par RNAi. Nous avons démontré que la phosphorylation de su(var)205 (S15) était modulée par l’insuline en plus d’être une cible directe de CK2 suite à l’expérience RNAi et à l’essai kinase. L’analyse des données phosphoprotéomiques a mis en évidence des phosphopeptides isomériques dont certains étaient séparables par FAIMS. Nous avons déterminé leur fréquence lors d’études à grande échelle grâce à deux algorithmes. Le script basé sur les différences de temps de rétention entre isomères a identifié 64 phosphoisomères séparés par LC chez la souris et le rat (moins de 1 % des peptides identifiés). Chez la drosophile, 117 ont été répertoriés en combinaison avec une approche ciblée impliquant des listes d’inclusion. Le second algorithme basé sur la présence d’ions caractéristiques suite à la fragmentation de formes qui co-éluent a rapporté 23 paires isomériques. L’importance de pouvoir distinguer des phosphoisomères est capitale dans le but d’associer une fonction biologique à un site de phosphorylation précis qui doit être identifié avec confiance. / Phosphorylation is a reversible post-translational modification that modulates protein activity, and can impart conformational changes and affect translocation of their protein substrates. Kinases and phosphatases are responsible for the dynamic of changes in protein phosphorylation and act in a coordinated manner. Abnormal activation or misregulation of kinase activity can lead to the development of cancers and metabolic disorders. Tyrosine kinase receptor (RTK) associated signaling pathways are often implicated in numerous diseases and the further understanding of mechanisms affecting their regulation is necessary to determine their activity and effects anticipated on their substrates. In this context, the primary objective of this thesis is to study the phosphorylation events arising from the activation of the insulin receptor (InR) following stimulation of drosophila S2 cells with insulin. The phosphorylation cascade triggered after InR activation is conserved in mammals. In order to study the phosphoproteome of drosophila S2 cells, we enriched phosphopeptides on titanium dioxide (TiO2) stationary phase prior to their separation by liquid chromatography (LC) and ion mobility (FAIMS) mass spectrometry (MS). Phosphopeptides were then analysed by tandem MS at high resolution. We first compared the benefits of FAIMS to conventional LC-MS, and observed a 50% increase in the number of identified phosphopeptides when using ion mobility. FAIMS enables the separation of phosphoisomers that are typically unresolved by LC, enabling high confidence assignment of modification sites via distinct MS/MS spectra. This approach was used to profile phosphorylation changes taking place between control and insulin-treated drosophila cells and enabled the identification of 32 phosphopeptides (out of 2 660 quantified) showing differential regulation. Interestingly, 50% of the regulated targets have a CK2 consensus site. These preliminary experiments were followed-up by RNAi mediated inhibition of CK2 and revealed that 6 phosphoproteins (CG30085, su(var)205, scny, protein CDV3 homolog, D1 and mu2) were positively modulated after insulin stimulation and negatively regulated after CK2 RNAi treatment. Using in vitro kinase assay, we identified 29 direct CK2 targets, of which 15 were correlated with results from the CK2 RNAi experiment. We demonstrated specifically that the su(var)205 (S15) is regulated by insulin and is a direct CK2 target based on RNAi and kinase assays. Our phosphoproteomics data also highlighted the presence of isomeric phosphopeptides, several of which could be distinguished using FAIMS. We developed two algorithms to determine the occurrence of phosphoisomers in large scale studies. The first algorithm based on differences in retention times between isomers identified 64 candidates in mouse and rat phosphoproteome datasets corresponding to less than 1% of all identified phosphopeptides. We also identified 117 isomer candidates in drosophila using a targeted LC-MS/MS approach with inclusion lists. The second algorithm is based on the presence of characteristic fragment ions present in MS/MS spectra of co-eluting or partially resolved species and allowed the identification of 23 isomeric pairs. The ability to distinguish phosphoisomers in large-scale phosphoproteome datasets is of significance to correlate phosphorylation events taking place on specific residues with biological activities.

La phosphorylation est une modification post-traductionnelle modulant l’activité, la conformation ou la localisation d’une protéine et régulant divers processus. Les kinases et phosphatases sont responsables de la dynamique de phosphorylation et agissent de manière coordonnée. L’activation anormale ou la dérégulation de kinases peuvent conduire au développement de cancers ou de désordres métaboliques. Les récepteurs tyrosine kinase (RTKs) sont souvent impliqués dans des maladies et la compréhension des mécanismes régissant leur régulation permet de déterminer les effets anticipés sur leurs substrats. Dans ce contexte, le but de cette thèse est d’identifier les évènements de phosphorylation intervenant dans la voie de l’insuline chez la drosophile impliquant un RTK : le récepteur de l’insuline (InR). La cascade de phosphorylation déclenchée suite à l’activation du récepteur est conservée chez le mammifère. Afin d’étudier le phosphoprotéome de cellules S2 de drosophile, nous avons utilisé une étape d’enrichissement de phosphopeptides sur dioxyde de titane suivie de leur séparation par chromatographie liquide (LC) et mobilité ionique (FAIMS). Les phosphopeptides sont analysés par spectrométrie de masse en tandem à haute résolution. Nous avons d’abord démontré les bénéfices de l’utilisation du FAIMS comparativement à une étude conventionnelle en rapportant une augmentation de 50 % dans le nombre de phosphopeptides identifiés avec FAIMS. Cette technique permet de séparer des phosphoisomères difficilement distinguables par LC et l’acquisition de spectres MS/MS distincts où la localisation précise du phosphate est déterminée. Nous avons appliqué cette approche pour l’étude des phosphoprotéomes de cellules S2 contrôles ou traitées à l’insuline et avons identifié 32 phosphopeptides (sur 2 660 quantifiés) pour lesquels la phosphorylation est modulée. Étonnamment, 50 % des cibles régulées possèdent un site consensus pour la kinase CK2. Une stratégie d’inhibition par RNAi a été implémentée afin d’investiguer le rôle de CK2 dans la voie de l’insuline. Nous avons identifié 6 phosphoprotéines (CG30085, su(var)205, scny, protein CDV3 homolog, D1 et mu2) positivement régulées suite à l’insuline et négativement modulées après le traitement par RNAi CK2. Par essai kinase in vitro, nous avons identifié 29 cibles directes de CK2 dont 15 corrélaient avec les résultats obtenus par RNAi. Nous avons démontré que la phosphorylation de su(var)205 (S15) était modulée par l’insuline en plus d’être une cible directe de CK2 suite à l’expérience RNAi et à l’essai kinase. L’analyse des données phosphoprotéomiques a mis en évidence des phosphopeptides isomériques dont certains étaient séparables par FAIMS. Nous avons déterminé leur fréquence lors d’études à grande échelle grâce à deux algorithmes. Le script basé sur les différences de temps de rétention entre isomères a identifié 64 phosphoisomères séparés par LC chez la souris et le rat (moins de 1 % des peptides identifiés). Chez la drosophile, 117 ont été répertoriés en combinaison avec une approche ciblée impliquant des listes d’inclusion. Le second algorithme basé sur la présence d’ions caractéristiques suite à la fragmentation de formes qui co-éluent a rapporté 23 paires isomériques. L’importance de pouvoir distinguer des phosphoisomères est capitale dans le but d’associer une fonction biologique à un site de phosphorylation précis qui doit être identifié avec confiance. / Phosphorylation is a reversible post-translational modification that modulates protein activity, and can impart conformational changes and affect translocation of their protein substrates. Kinases and phosphatases are responsible for the dynamic of changes in protein phosphorylation and act in a coordinated manner. Abnormal activation or misregulation of kinase activity can lead to the development of cancers and metabolic disorders. Tyrosine kinase receptor (RTK) associated signaling pathways are often implicated in numerous diseases and the further understanding of mechanisms affecting their regulation is necessary to determine their activity and effects anticipated on their substrates. In this context, the primary objective of this thesis is to study the phosphorylation events arising from the activation of the insulin receptor (InR) following stimulation of drosophila S2 cells with insulin. The phosphorylation cascade triggered after InR activation is conserved in mammals. In order to study the phosphoproteome of drosophila S2 cells, we enriched phosphopeptides on titanium dioxide (TiO2) stationary phase prior to their separation by liquid chromatography (LC) and ion mobility (FAIMS) mass spectrometry (MS). Phosphopeptides were then analysed by tandem MS at high resolution. We first compared the benefits of FAIMS to conventional LC-MS, and observed a 50% increase in the number of identified phosphopeptides when using ion mobility. FAIMS enables the separation of phosphoisomers that are typically unresolved by LC, enabling high confidence assignment of modification sites via distinct MS/MS spectra. This approach was used to profile phosphorylation changes taking place between control and insulin-treated drosophila cells and enabled the identification of 32 phosphopeptides (out of 2 660 quantified) showing differential regulation. Interestingly, 50% of the regulated targets have a CK2 consensus site. These preliminary experiments were followed-up by RNAi mediated inhibition of CK2 and revealed that 6 phosphoproteins (CG30085, su(var)205, scny, protein CDV3 homolog, D1 and mu2) were positively modulated after insulin stimulation and negatively regulated after CK2 RNAi treatment. Using in vitro kinase assay, we identified 29 direct CK2 targets, of which 15 were correlated with results from the CK2 RNAi experiment. We demonstrated specifically that the su(var)205 (S15) is regulated by insulin and is a direct CK2 target based on RNAi and kinase assays. Our phosphoproteomics data also highlighted the presence of isomeric phosphopeptides, several of which could be distinguished using FAIMS. We developed two algorithms to determine the occurrence of phosphoisomers in large scale studies. The first algorithm based on differences in retention times between isomers identified 64 candidates in mouse and rat phosphoproteome datasets corresponding to less than 1% of all identified phosphopeptides. We also identified 117 isomer candidates in drosophila using a targeted LC-MS/MS approach with inclusion lists. The second algorithm is based on the presence of characteristic fragment ions present in MS/MS spectra of co-eluting or partially resolved species and allowed the identification of 23 isomeric pairs. The ability to distinguish phosphoisomers in large-scale phosphoproteome datasets is of significance to correlate phosphorylation events taking place on specific residues with biological activities.

Groundwater aquifers are the largest source of drinking water for human population. Current available information of the quality of groundwater is quite limited mainly due to the lack of comprehensive analysis of groundwater and the challenging task of applying any analytical method in its investigation. In this thesis, a new method based on “soft” mass spectrometry and differential ion mobility (FAIMS) was developed to discover previously unknown sulphur-containing contaminants in groundwater in Ontario. Following this discovery, de novo identification of these contaminants was accomplished by determining their elemental composition based on mass measurements and their chemical structures from unique dissociation patterns. The compounds characterized in this study were found to be thiotetronic acids which are structurally related to synthetic and natural antibacterial agents such as the natural antibiotics thiolactomycin and thiotetramycin, allowing for speculation as to their potential beneficial properties.

Identification of trace unknown analytes in complex samples remains a significant challenge for analytical chemistry. Mass spectrometry (MS) and analytical separations techniques can now be used to develop and support a new analytical strategy called non-target analysis which aims to provide comprehensive identification and quantification of all detectable chemical species in a complex sample. This thesis addresses challenges currently limiting the utility of this non-target approach by developing analytical methods for acquiring MS data suitable for identification of trace unknowns and investigating current tools available for unknown identification from MS spectral data. Liquid chromatography (LC) - MS, a widely used technique in trace analysis, was used to develop an analytical method capable of simultaneously acquiring high resolution MS and tandem mass spectrometry (MS/MS) data for hundreds of metabolites in urine. An emerging separation technique called high field asymmetric waveform ion mobility spectrometry (FAIMS) was also investigated, as an alternative to LC, for the identification of non-target analytes in urine. Modifications were carried out to the FAIMS-MS source interface allowing for transmission of small metabolite ions from FAIMS to MS. The challenge of direct electrospray (ESI) in urine analysis using ESI-FAIMS-MS was addressed by using sample dilution and extending MS data acquisition time using FAIMS. This allowed for higher quality MS data to be acquired for low abundance urinary metabolites than was possible by LC-MS and the complete elimination of ionization suppression in dilute urine samples. Insight gained into ESI suppression in complex samples allowed for two methods of semi-quantification to be proposed for non-target analytes in complex samples without using unavailable chemical standards. To address the challenge of unknown identification, faced throughout this thesis, an integrated approach was implemented to identify metabolites based only on spectral data without the usual requirement of availability of chemical standards. This approach combined spectral libraries, literature reports on ion chemistry and de novo identification based on gas phase ion chemistry with a detailed fragmentation study on nucleic acid bases, notably protonated uracil. Together, the instrumental methods and approaches to data analysis described allowed for the identification of 110 abundant chemical species detected in urine. / Natural Sciences and Engineering Research Council of Canada, Ontario Ministry of Training, Colleges and Universities, Canadian Foundation for Innovation

La grande complexité des échantillons biologiques peut compliquer l'identification des protéines et compromettre la profondeur et la couverture des analyses protéomiques utilisant la spectrométrie de masse. Des techniques de séparation permettant d’améliorer l’efficacité et la sélectivité des analyses LC-MS/MS peuvent être employées pour surmonter ces limitations. La spectrométrie de mobilité ionique différentielle, utilisant un champ électrique élevé en forme d'onde asymétrique (FAIMS), a montré des avantages significatifs dans l’amélioration de la transmission d'ions peptidiques à charges multiples, et ce, en réduisant les ions interférents. Dans ce contexte, l'objectif de cette thèse était d'explorer les capacités analytiques de FAIMS afin d'élargir à la fois la gamme dynamique de détection des protéines/peptides et la précision des mesures en protéomique quantitative par spectrométrie de masse. Pour cela, nous avons systématiquement intégré FAIMS dans des approches classiques en protéomique afin de déterminer les changements dynamiques du protéome humain en réponse à l’hyperthermie. Nous avons d’abord étudié les avantages de FAIMS par rapport à la quantification par marquage isobare (tandem mass tag, TMT). Cette approche permet le marquage d'ions peptidiques avec différents groupements chimiques dont les masses nominales sont identiques mais différant par leur distribution respective d'isotopes stables. Les ions peptidiques marqués par TMT produisent des ions rapporteurs de masses distinctes une fois fragmentés en MS/MS. Malheureusement, la co-sélection d'ions précurseurs conduit souvent à des spectres MS/MS chimériques et une approche plus lente basée sur le MS3 est nécessaire pour une quantification précise. Comme FAIMS améliore l’efficacité de séparation en transmettant sélectivement des ions en fonction de leur voltage de compensation (CV), nous avons obtenu moins de co-sélection de peptides. FAIMS a amélioré la quantification des peptides TMT au niveau MS2 et a permis d’obtenir 68% plus de peptides quantifiés par rapport aux analyses LC-MS/MS classiques, fournissant ainsi un aperçu plus vaste des changements dynamiques du protéome humain en réponse au stress thermique. De plus, nous avons étudié le marquage métabolique par incorporation d’acides aminés marqués par des isotopes stables en culture cellulaire (SILAC). Si des interférences co-éluent avec les isotopes SILAC, la quantification devient imprécise et les contreparties de SILAC peuvent être assignées de manière erronée aux ions interférants du chromatogramme, faussant ainsi le rapport SILAC. Le fractionnement post-ionisation FAIMS pourrait filtrer les ions appartenant au bruit de fond qui pourraient autrement être attribués à une paire ou à un triplet SILAC pour la quantification. Dans ce projet, FAIMS a été particulièrement bénéfique pour les espèces peu abondantes et s’est montré plus performant que le fractionnement par échange de cations (SCX). En outre, FAIMS a permis la séparation des phosphoisomères fréquemment observés dans les extraits complexes de phosphoprotéomes. Le troisième objectif de ce travail de recherche était d'explorer la séparation de l'état de charge et la transmission améliorée de peptides fortement chargés avec FAIMS et son application à l'analyse de peptides SUMOylés. FAIMS pourrait ainsi améliorer la transmission des peptides SUMOylés triplement chargés par rapport aux peptides tryptiques usuels, lesquels sont principalement doublement chargés. Ceci permettait l'enrichissement en phase gazeuse des ions peptides SUMOylés. FAIMS est une approche alternative plus simple pour fractionner les peptides SUMOylés, ce qui réduit les pertes d’échantillon et permet de simplifier le traitement des échantillons, tout en augmentant l’efficacité de séparation de manière plus automatisée et en ajoutant un ordre de grandeur de sensibilité. Le dernier objectif de cette thèse était d’améliorer l’instrumentation de FAIMS en le jumelant aux instruments à la fine pointe de la technologie. Avec un nouveau dispositif FAIMS, développé par nos collaborateurs chez Thermo Fisher Scientific, nous avons montré une amélioration dans la robustesse et la transmission des ions pour la nouvelle interface. Dans des expériences simples en protéomique shotgun, FAIMS a étendu la gamme dynamique d'un ordre de grandeur pour une couverture protéomique plus profonde par rapport aux analyses LC-MS/MS classiques. En outre, le fractionnement en phase gazeuse de FAIMS a généré moins d’analyses chimériques en MS2, ce qui a permis d’obtenir plus d’identifications et une meilleure quantification. Pour ce faire, nous avons directement comparé le LC-FAIMS-MS/MS au LC-MS/MS/MS en utilisant la sélection de précurseur synchrone (SPS) avec et sans fractionnement en phase inverse basique. Des mesures quantitatives comparables ont été obtenues pour toutes les méthodes, à l'exception du fait que FAIMS a parmi d’obtenir un nombre 2,5 fois plus grand de peptides quantifiables par rapport aux expériences sans FAIMS. Globalement, cette thèse met en évidence certains des avantages que FAIMS peut offrir aux expériences en protéomique en améliorant à la fois l'identification et la quantification des peptides. / 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.

La sensibilité et la profondeur de l'analyse protéomique sont limitées par les ions isobares et les interférences qui entravent l'identification des peptides de faible abondance. Lorsque nous analysons des échantillons de grande complexité, une séparation extensive de l'échantillon est souvent nécessaire pour étendre la couverture protéomique. Ces dernières années, la spectrométrie de mobilité ionique à forme d'onde asymétrique à haut champ (FAIMS) a gagné en popularité dans le domaine de la protéomique pour sa capacité à séparer les ions isobares, à améliorer la capacité de pic et la sensibilité de la spectrométrie de masse (MS). Nous rapportons ici l'intégration d'un appareil FAIMS Pro™ à un Q-Exactive HF™ ainsi qu'un spectromètre de masse Orbitrap Exploris 480™. Des expériences protéomiques sur des digestions d'extraits protéiques issues de cellules Hela à l'aide d'un spectromètre de masse avec FAIMS ont amélioré le rapport signal sur bruit (S/N) et réduit les ions interférents, ce qui a entraîné une augmentation du taux d'identification des peptides de plus de 42 %. FAIMS est également combiné avec le fractionnement ionique segmenté (SIFT), qui utilise tour à tour une fenêtre de 100 ~ 300 m/z au lieu de la large plage traditionnelle (700 ~ 800 m/z), augmentant ainsi la profondeur de la couverture protéomique tout en réduisant la proportion de spectres MS/MS chimériques de 50% à 27%. Dans l'analyse quantitative, nous démontrons l'application de FAIMS pour améliorer les mesures quantitatives lorsque le marquage peptidique isobare est utilisé. Par rapport aux expériences LC-MS/MS conventionnelles, la combinaison des expériences FAIMS et SIFT réalisées sur un modèle à deux protéomes a montré une amélioration de 65 % de la précision des mesures quantitatives. Les digestions tryptiques d'extraits protéiques de différentes lignées cellulaires du cancer colorectal ont été utilisées pour l'évaluation de stratégie combinée FAIMS et SIFT sur un spectromètre de masse Orbitrap Exploris 480™ offre un gain d'identification de 70 % par rapport à l'approche conventionnelle et combinée aux données transcriptomiques elle facilite l’identification de variants protéiques. / The sensitivity and depth of proteomic analysis in mass spectrometry (MS) is limited by isobaric ions and interferences that hinder the identification of low-abundance peptides. For high complexity samples, extensive separation is often required to expand proteomic coverage. In recent years, high-field asymmetric waveform ion mobility spectrometry (FAIMS) has gained popularity in the field of proteomics for its ability to resolve confounding ions, improve peak capacity, and sensitivity. This thesis presents the integration of a FAIMS Pro™ interface with electrical and gas embedded connections to a Q-Exactive HF™ as well as an Orbitrap Exploris 480™ mass spectrometer. Proteomic experiments on tryptic digests of HeLa cell line using a FAIMS integrated mass spectrometer improved signal-to-noise ratio (S/N) and reduced the occurrence of interfering ions. This enabled a 42% increase in peptide identification rate. Also, FAIMS was combined with segmented ion fractionation (SIFT), which in turn scans with windows of 100~300 m/z width instead of the traditional width (700~800 m/z), further increasing the depth of proteome coverage by a reducing from 50% to 27% in terms of MS/MS chimeric spectra numbers. The application of FAIMS gain improvement on quantitative measurements with TMT labeling method is presented. Compared to conventional LC-MS/MS tests, the combination of FAIMS and SIFT experiments showed a improvement by 65% in quantitative accuracy when performed on a human-yeast two-proteome model. As an application of the method, the tryptic digests from different colorectal cancer cell lines were used for the evaluation. FAIMS-SIFTcombined strategy on an Orbitrap Exploris 480™ mass spectrometer provides a 70% gain in identification compared to the conventional LC-MS/MS approach for the same sample amount and instrument time. This enhanced sensitivity facilitates single amino acid mutations confirmed by RNAseq analyses.