Proteome characterizations of microbial systems using MS-based experimental and informatics approaches to examine key metabolic pathways, proteins of unknown function, and phenotypic adaptation

Microbes express complex phenotypes and coordinate activities to build microbial communities. Recent work has focused on understanding the ability of microbial systems to efficiently utilize cellulosic biomass to produce bioenergy-related products. In order to maximize the yield of these bioenergy-related products from a microbial system, it is necessary to understand the molecular mechanisms.The ability of mass spectrometry to precisely identify thousands of proteins from a bacterial source has established mass spectrometry-based proteomics as an indispensable tool for various biological disciplines. This dissertation developed and optimized various proteomics experimental and informatic protocols, and integrated the resulting data with metabolomics, transcriptomics, and genomics in order to understand the systems biology of bio-energy relevant organisms. Integration of these various omics technologies led to an improved understanding of microbial cell-to-cell communication in response to external stimuli, microbial adaptation during deconstruction of lignocellulosic biomass and proteome diversity when an organism is subjected to different growth conditions.Integrated omics revealed Clostridium thermocellum\u27s accumulate long-chain, branched fatty acids over time in response to cytotoxic inhibitors released during the deconstruction and utilization of switchgrass. A striking feature implies a restructuring of C. thermocellum\u27s cellular membrane as the culture progresses. The membrane remodulation was further examined in a study involving the swarming and swimming phenotypes of Paenibacillus polymyxa. The possible roles of phospholipids, hydrolytic enzymes, surfactin, flagellar assembly, chemotaxis and glycerol metabolism in swarming motility were investigated by integrating lipidomics with proteomics.Extracellular proteome analysis of Caldicellulosiruptor bescii revealed secretome plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. This study further opened the avenue for research to characterize proteins of unknown function (PUFs) specific to growth conditions.To gain a better understanding of the possible functions of PUFs in C. thermocellum, a time course analysis of C. thermocellum was conducted. Based on the concept of guilt-by-association, protein intensities and their co-expressions were used to tease out the functional aspect of PUFs. Clustering trends and network analysis were used to infer potential functions of PUFs. Selected PUFs were further interrogated by the use of phylogeny and structural modeling

Performance-Based Quality Specifications: The Link between Product Development and Clinical Outcomes

The design of drug delivery systems and their corresponding dosing guidelines are critical product development functions supported by clinical pharmacokinetic (PK) and pharmacodynamic (PD) data. Largely, the importance of variance and covariance in product and patient attributes is poorly understood. The existence of PK/PD diversity among myriad patient sub-populations further complicates efforts to gauge the importance of product quality variation. Nevertheless, a platform capable of evaluating the effects of product and patient variability on clinical performance was constructed. This dissertation was predicated on requests to re-define pharmaceutical quality in terms of risk by relating clinical attributes to production characteristics. To avoid in vivo studies, simulated experimental trials were conducted using the model drug, theophylline, for which data and models could be acquired from the literature. Where comprehensive data were unavailable (e.g., production variability statistics), initial estimates were acquired via laboratory-scale experiments. Model asthmatic patients were generated using Monte Carlo simulation and published population distributions of various anothropometric measurements, disease rates, and lifestyle factors. Mathematical constructs for in vitro-in vivo correlations provide a linkage between Quality by Design (QbD) product and process models, PK/PD models, and patient population statistics. The combined models formed the foundation for Monte Carlo risk assessments, which characterized the risk of inefficacy and toxicity for dosing of extended-release theophylline tablets. Sensitivity analyses revealed that patient compliance and content uniformity significantly influenced the probability of observing an adverse event. The Monte Carlo risk assessment platform defined the link between the critical quality attributes (CQAs) and clinical performance (i.e., performance-based quality specifications (PBQS)). The PBQS were subsequently utilized to generate process independent design spaces conditioned on inefficacy and toxicity risk. These design spaces, which directly account for the conditional relationships between product quality and patient variability, can be transferred to a specific process via models that relate process critical control parameters to the CQAs. Process Analytical Technology, therefore, can be integrated into the QbD production environment to control the safety and efficacy of the final product. This work demonstrated that process and product knowledge can be used to estimate the risk that final product quality imparts to clinical performance

Non-invasive biochemical analysis of cells, tissues and tissue constructs with Raman micro-spectroscopy

Imperial Users onl

An integrated proteomic and metabolomic approach to investigate cerebral ischemic preconditioning

The molecular mechanism that leads to ischemic preconditioning and hence to ischemic tolerance, are not completely understood although it is clear that multiple effectors and pathways contribute to the instauration of this neuroprotective profile. To study the mechanism/pathway involved in the ischemic tolerance, brain proteins, plasma proteins and plasma metabolites were analyzed in preconditioning stimulus (7'Middle Cerebral Artery occlusion or 7'MCAo), in severe stroke and (permanent Middle Cerebral Artery occlusion or pMCAo) and in preconditioned (7'MCAo/pMCAo) mouse model. A conventional 2-DE approach was used to study technical replicates of pooled brain proteins revealing an involvement of energy metabolism, mitochondrial electron transport, synaptic vesicle transport and antioxidant processes; moreover network analysis suggested an involvement of the androgen receptor that was validated on technical replicates of pooled brain proteins by western blot analysis revealing an increased expression in preconditioned stimulus animals (7'MCAo). Plasma proteins were analyzed using a i-DE LC-MS/MS approach on technical replicates of pooled plasma proteins revealing decreased levels of epidermal growth factor receptor (EGFR) and increased levels of insuline like growth factor acid labile subunit (IGFALS), which expression was paralleled by increased insulin like growth factor 1 (IGFi) plasma concentration, as validated by ELISA on biological replicates, in preconditioning stimulus animals (7'MCAo). Finally an untarget metabolomics analysis was applied to technical replicates of pooled plasma proteins revealing fatty acid oxidation and branched-chain aminoacid metabolism as the main biological processes modulated in ischemic tolerance and highlighted an involvement of the aminoacid leucine, carnitine esters and adenosine. The results described in this thesis represents the first application of both proteomic and metabolomic approaches in cerebral ischemic sets, highlighting the androgen receptor as an important mediator between proteins and metabolites and adding new evidence to the current knowledge on ischemic preconditioning that may represent a starting point for future experiments on investigating candidate pathways that relate to the Androgen receptor

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

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

Incorporating standardised drift-tube ion mobility to enhance non-targeted assessment of the wine metabolome (LC×IM-MS)

Liquid chromatography with drift-tube ion mobility spectrometry-mass spectrometry (LCxIM-MS) is emerging as a powerful addition to existing LC-MS workflows for addressing a diverse range of metabolomics-related questions [1,2]. Importantly, excellent precision under repeatability and reproducibility conditions of drift-tube IM separations [3] supports the development of non-targeted approaches for complex metabolome assessment such as wine characterisation [4]. In this work, fundamentals of this new analytical metabolomics approach are introduced and application to the analysis of 90 authentic red and white wine samples originating from Macedonia is presented. Following measurements, intersample alignment of metabolites using non-targeted extraction and three-dimensional alignment of molecular features (retention time, collision cross section, and high-resolution mass spectra) provides confidence for metabolite identity confirmation. Applying a fingerprinting metabolomics workflow allows statistical assessment of the influence of geographic region, variety, and age. This approach is a state-of-the-art tool to assess wine chemodiversity and is particularly beneficial for the discovery of wine biomarkers and establishing product authenticity based on development of fingerprint libraries

Integrating glycomics, proteomics and glycoproteomics to understand the structural basis for influenza a virus evolution and glycan mediated immune interactions

Glycosylation modulates the range and specificity of interactions among glycoproteins and their binding partners. This is important in influenza A virus (IAV) biology because binding of host immune molecules depends on glycosylation of viral surface proteins such as hemagglutinin (HA). Circulating viruses mutate rapidly in response to pressure from the host immune system. As proteins mutate, the virus glycosylation patterns change. The consequence is that viruses evolve to evade host immune responses, which renders vaccines ineffective. Glycan biosynthesis is a non-template driven process, governed by stoichiometric and steric relationships between the enzymatic machinery for glycosylation and the protein being glycosylated. Consequently, protein glycosylation is heterogeneous, thereby making structural analysis and elucidation of precise biological functions extremely challenging. The lack of structural information has been a limiting factor in understanding the exact mechanisms of glycan-mediated interactions of the IAV with host immune-lectins. Genetic sequencing methods allow prediction of glycosylation sites along the protein backbone but are unable to provide exact phenotypic information regarding site occupancy. Crystallography methods are also unable to determine the glycan structures beyond the core residues due to the flexible nature of carbohydrates. This dissertation centers on the development of chromatography and mass spectrometry methods for characterization of site-specific glycosylation in complex glycoproteins and application of these methods to IAV glycomics and glycoproteomics. We combined the site-specific glycosylation information generated using mass spectrometry with information from biochemical assays and structural modeling studies to identify key glycosylation sites mediating interactions of HA with immune lectin surfactant protein-D (SP-D). We also identified the structural features that control glycan processing at these sites, particularly those involving glycan maturation from high-mannose to complex-type, which, in turn, regulate interactions with SP-D. The work presented in this dissertation contributes significantly to the improvement of analytical and bioinformatics methods in glycan and glycoprotein analysis using mass spectrometry and greatly advances the understanding of the structural features regulating glycan microheterogeneity on HA and its interactions with host immune lectins

Novel Engineering Approaches for DNA Sequencing and Analysis

DNA sequencing is a fundamental tool in biological and medical research. DNA molecules contain the heritable genetic information in all living organisms and encode all the proteins in our body. Therefore, determination of DNA sequence is useful in basic biological research, evolutionary biology, as well as the applied biological fields, such as diagnostic or forensic research. High-throughput DNA sequencing is essential for personalized medicine. To achieve this dream, the price of genome sequencing should be dramatically decreased to a level that most people can afford. Despite the refinements of Sanger sequencing, the current genome sequencing cost remains formidable. Therefore, revolutionary advances in DNA sequencing technology are demanded. To overcome the limitations of the current sequencing technologies, a variety of new DNA sequencing methods have been investigated with the aim of eventually realizing the goal of the $1,000 genome, including sequencing by synthesis (SBS). In this thesis, we build upon current state-of-the-art sequencing technologies such as SBS to develop novel proof-of-principle technologies for high-throughput DNA sequencing; demonstrate a general platform for high sensitivity biomolecular detection; and briefly study DNA processing protein (e.g., helicase) functions at the atomistic level. The following is a summary of the resulting work presented herein: first, a new DNA sequencing technology development is presented that utilizes surface-enhanced Raman spectroscopy (SERS); second, a novel approach of SERS-based biosensing for quantitative detection of biomolecules is demonstrated; third, a rigorous mathematical development of an analytical model is described to predict experimental SERS signal intensity distributions for biomolecular quantification; fourth, a versatile SERS-based quantitative method is developed to monitor the catalyst-free click reaction efficiency for small molecule conjugation; fifth, theoretical work using molecular dynamics simulations for analyzing the mechanical behavior of a molecular motor involved in DNA processing are described; and finally, the thesis presents a proposed nanodevice to combine the SERS-SBS technology with our bioquantification method into one functional unit. Consequently, these research efforts provide a foundation for the novel use and integration of SERS-SBS into microfluidic systems for a wide range of applications, such as high-throughput DNA sequencing and genetic diagnostics, as well as the theoretical framework to investigate DNA polymerase function at the atomic level