Structure determination of macromolecular protein assemblies remains a challenge for well-established experimental methods. In this thesis, an emerging structural technique, ion mobility-mass spectrometry (IM-MS) is explored. An assessment of collision cross section (CCS) measurement accuracy using travelling-wave IM (TWIMS) instrumentation was carried out (Chapter 3). Through the collation of a protein complex CCS database and the development of a calibration framework for TWIMS, significant improvements to CCS measurement accuracy have been achieved. Next, the advantages and limitations of using IM-MS to generate restraints for structure characterisation were explored. Computational tools designed to exploit IM-MS data for structural modelling were developed and tested on a training set of systems (Chapter 4). These include two heteromeric protein complexes, and an oligomeric intermediate involved in beta-2-microglobulin aggregation. Further structural information can be attained by using gas-phase dissociation techniques, such as collision-induced dissociation (CID). The effects of charge state on CCS and the gas-phase dissociation pathway of complexes were investigated (Chapter 5). This highlighted the possibility of using CID in conjunction with supercharging to manipulate dissociation pathways to achieve more useful structural information. Finally, the gas-phase structures of globular and intrinsically disordered protein complexes were probed by IM-MS and molecular dynamics (MD) simulations (Chapter 6). Experimental observations were recapitulated remarkably closely by simulations, including gas-phase structural collapse and the ejection of monomer subunits when the energy of the system was increased sufficiently. Overall, this research has contributed to the IM-MS field by providing the framework for improved CCS measurements of large protein complexes and the use of restraints from IM-MS for structural modelling. Significantly, IM-MS has been used in combination with charge manipulation, CID and MD simulations to reveal further insights into the gas-phase structures, stabilities and dissociation pathways of multimeric protein complexes.

This dissertation presents efforts to expand the role of mass spectrometry (MS) in structural biology. Determination of quaternary structure of a protein complex has been hindered by limited fragmentation from collision-induced dissociation (CID). As an alternative, surface-induced dissociation (SID) was implemented for a quadrupole - time-of-flight instrument in the Wysocki laboratory. This research tested the hypothesis that SID should produce fragmentation reflective of subunit organization. Furthermore, ion mobility (IM) was used to prove the direct relationship between precursor conformation and observed dissociation patterns, and the relationship between activation and product ion conformation. The structure and dynamics of a dimeric small heat shock protein (sHSP) with no solved structure was investigated. The importance of N- and C-terminal domains for dimerization was determined, and the dimers were shown to exchange subunits. From exchange kinetics it is proposed that subunit exchange is unrelated to heat shock activity. SID was used to elucidate the subunit architecture of heterogeneous protein assemblies, including one previously solved protein structure and two formerly uncharacterized proteins. The heterohexamer toyocamycin nitrile hydratase dissociated into trimers, revealing the hexamer to be a dimer of trimers. The bacterial ribonuclease toxin:antitoxin tetramer was shown to have an antitoxin dimer at its core, with flanking individual toxin subunits. The examples presented here are the first clear proof that SID results can clearly indicate the substructure of a protein assembly.IM was used to study the conformation of precursor and product ions. A greater understanding of the relationship between precursor conformation and observed dissociation patterns was developed. Different charge states of a dodecameric sHSP were found to have significantly different conformations, which were directly reflected in SID spectra. IM comparison of CID and SID product ions showed that the same charge state of a product ion from either method has the same CCS. This suggests the product ion conformation is dependent upon ion charge state, and independent of activation method and collision energy. The cause and effect relationship between precursor conformation and MS/MS patterns, and activation and product ion conformation were clearly illustrated. Together, this body of research expands the role of MS for structural biology.

Biological systems of bacteria were investigated at the single-cell and single-molecule level. Additionally, aspects of the techniques employed were studied. A unifying theme in each project is the reliance on optical imaging techniques coupled to microfluidic devices. Hypo-osmotic shock experiments with an Escherichia coli mechanosensitive channel deletion mutant were carried out at the single-cell level. E. coli MJF465 cells in which the three major mechanosensitive channel genes are deleted (∆mscL, ∆mscS, ∆mscK) show only 10% cell viability upon hypo-osmotic shock (from LB + 0.5 M NaCl into distilled water), compared to 90% viability of the wild-type strain. Bacterial cells were trapped with optical tweezers in microfluidic devices, enabling the first direct observation of single-cell behaviour upon hypo-osmotic shock. Phase-contrast microscopy revealed intra-population diversity in the cells response: Different features of lysis included cells bursting rapidly and leakage of ribosomes, DNA and protein from the cytoplasm. Fluorescence microscopy of hypo-osmotically-shocked GFP-expressing MJF465 cells showed either bursting of cells, which was a rare event, or fast leakage of GFP, indicating cell membrane ruptures. Data were analysed in terms of their kinetic behaviour and showed that lysis occurs on a timescale of milliseconds to seconds. The implications of these findings for the bacterial cell wall and cell membranes are discussed. Enzymes involved in homologous recombination and repair of double-stranded DNA (dsDNA) breaks are essential for maintaining genomic integrity in both eukaryotes and prokaryotes. RecBCD of E. coli and AddAB, found widely in bacteria, are involved in these processes, carrying out the same function. Both enzymes were studied kinetically with single-molecule total internal reflection fluorescence microscopy (TIRFM). Surface-tethered, hydrodynamically stretched lambda-DNA molecules, stained with YOYO-1, were imaged with TIRFM in a microfluidic flowcell. The RecBCD enzyme is a well characterised DNA helicase and was introduced to this system for method validation purposes. The AddAB enzyme of Bacteroides fragilis was then characterised as a helicase acting on lambda-DNA. It was found that AddAB helicase unwinds dsDNA with high processivity of on average 14,000 bp and up to 40,000 bp for individual enzyme complexes at an ATP-dependent rate ranging from 50-250 bp s−1 (for Mg2+-ATP concentrations larger or equal than 0.1 mM). This activity was detected by DNA binding dye (YOYO-1) displacement from the dsDNA and studied for different Mg2+-ATP concentrations, flow (shear) rates and different YOYO-1 staining ratios of DNA. Aspects of this last experimental setup were investigated. A kinetic analysis of intercalation of YOYO-1 into lambda-DNA is presented, occurring on a timescale of minutes. Different flow rates and staining ratios that influence the apparent (stretched) DNA molecule length were also examined. Several image analysis techniques were employed to enhance the data quality in images showing stretched lambda-DNA molecules. The Singular Value Decomposition was found to be the most effective technique which strongly reduces the noise in the obtained kymograph images.

High-throughput technology is changing the face of research biology, generating an ever growing amount of large-scale data sets. With experiments utilizing next-generation gene sequencing, mass spectrometry, and various other global surveys of proteins, the task of translating the plethora of data into biology has become a daunting task. In response, functional networks have been developed as a means for integrating the data into models of proteomic organization. In these networks, proteins are linked if they are evidenced to operate together in the same function, facilitating predictions about the functions, phenotypes, and disease associations of uncharacterized genes. In this body of work, we explore different applications of this so-called "guilt-by-association" concept to predict loss-of-function phenotypes and diseases associated with genes in yeast, worm, and human. We also scrutinize certain limitations associated with the functional networks, predictive methods, and measures of performance used in our studies. Importantly, the predictive method and performance measure, if not chosen appropriately for the biological objective at hand, can largely distort the results and interpretation of a study. These findings are incorporated in the development of RIDDLE, a method for characterizing whole sets of genes. This machine learning-based method provides a measure of network distance, and thus functional association, between two sets of genes. RIDDLE may be applied to a wide range of potential applications, as we demonstrate with several biological examples, including linking microRNA-450a to ocular development and disease. In the last decade, functional networks have proven to be a useful strategy for interpreting large-scale proteomic and genomic data sets. With the continued growth of genome coverage in networks and the innovation of predictive methods, we will surely advance towards our ultimate goal of understanding the genetic changes that underlie disease. / text

This dissertation presents an investigation into the structure of macromolecular protein assemblies by mass spectrometry. The experiments described within are designed to systematically assess the analytical utility of surface-induced dissociation (SID) tandem mass spectrometry in the characterization of multi-subunit protein complexes. This is accomplished by studying the effects of ion-surface collision on the fragmentation products of protein assemblies that vary by mass, number of subunits, and protein structural features. The dissociation energetics and mechanisms of protein complexes are considered by examining the influence of ion internal energy and sub-oligomeric protein structure on the dissociation process. Conditions are first established for the preservation of “native” protein quaternary structure and applied to previously characterized systems for proof-ofconcept. These conditions are subsequently extended to determine the molecular weight and subunit stoichiometry of several small heat shock proteins. Native mass spectrometry is then combined with limited proteolysis experiments to characterize the subunit interface of a unique small heat shock protein, Hsp18.5 from Arabidopsis thaliana, identifying regions of the protein essential for preservation of the native dimer. The dissociation of non-covalent protein assemblies is then explored on a quadrupole time-of-flight (Q-TOF) mass spectrometer, modified for the study of ion-surface collisions. This instrument allows ions to be dissociated through collisions with a surface or more conventional collisions with gas atoms. The dissociation of protein complexes is explored by both activation methods beginning with specific and non-specific dimers with masses less than 40 kDa. These studies are extended to larger assemblies with as many as 14 subunits weighing over 800 kDa, and are applied to both homo- and hetero-oligomeric protein complexes. Activation of a protein complex with “n” subunits through multiple collisions with inert gas atoms results in asymmetric dissociation into a highly charged monomer and complementary (n-1)-mer regardless of protein size or subunit architecture. This process is known to occur through an unfolding of the ejected subunit, and limits the amount of structural insight that can be gleaned from such studies. Collision at a surface however, results in more charge and mass symmetric fragmentation, and in some instances reflects the substructure of the protein assembly under investigation. The differences observed between the CID and SID of protein complexes is attributed to the rapid deposition of large amounts of internal energy deposited upon collision at a more massive target such as a surface. The ion activation time-frame and energy transfer efficiency are proposed to induce dissociation on a time-scale that precedes subunit unfolding providing access to dissociation pathways that are inaccessible by traditional means of activation. The systems studied here represent the largest ions fragmented via surface collisions within a mass spectrometer, and the fragmentation products observed by SID demonstrate its promise for expanding the role of mass spectrometry in the field of structural biology.

Proteins play important roles in most cellular functions, such as, replication, transcription regulation, signal transduction, for catalyzing chemical reaction, etc. Technologies developed to identify proteins rely either on observing their own properties such as charge, size, mass to charge ratio or sequence composition; or on using affinity reagents that recognize specific protein targets. Immunoassays utilizing functionalized affinity reagents are powerful for targeted proteomics. Among them, DNA-assisted immunoassays in which affinity reagents are labeled with DNA molecules, offer some unique advantages. In this thesis, I will present works to improve current DNA-assisted immunoassays such as proximity ligation assays (PLA), as well as to take advantage of DNA reactions to adress other problems. In paper I, a new solid support (MBC-Ts) was functionalized with antibodies and used in the solid-phase PLA for detection of VEGF. The assay using MBC-Ts was compared among the commercially available solid supports in different matrices and it was shown to exhibit enhanced limit of detection in complex matrices. In paper II, a two-step protocol was described to prepare high-quality probes used in homogeneous and in situ PLA by purifying DNA-labeled affinity reagents from unconjugated affinity reagents and excess oligonucleotides. In paper III, PLA was applied on a capillary western blotting instrument so that both the sensitivity and specificity of the original assay were improved. In paper IV, a new method was introduced to profile protein components in individual protein complexes by DNA-barcoded antibodies. This method has been used to profile protein complexes such as surface proteins on individual secreted vesicles.

No description available.

Pour comprendre comment les gènes du développement exercent de multiples fonctions temporelles, nous prenons comme modèle le facteur de transcription Otx2. Celui-ci est impliqué dans la gastrulation, le développement de l’œil, du système olfactif, de la glande pinéale, du thalamus et de la région cranio-faciale. Dans la rétine adulte, deux tissus distincts expriment Otx2 : l’épithélium pigmenté (RPE) et la rétine neurale, contenant les photorécepteurs. L’ablation globale du gène Otx2 entraîne la dégénérescence exclusive des photorécepteurs alors qu’elle modifie l’expression de gènes surtout dans le RPE. Ces faits suggèrent un mécanisme non autonome, confirmé par des expériences de gain et perte de fonction restreintes au RPE. Pour approcher les fonctions de la protéine Otx2 dans la rétine neurale et le RPE, une étude à grande échelle de ses cibles génomiques a été menée. Les profils distincts d’occupation du génome du RPE et de la rétine neurale suggèrent des fonctions différentes d’Otx2. Dans la rétine neurale, ce profil est très proche de celui du facteur paralogue Crx, indiquant une redondance fonctionnelle entre Otx2 et Crx. Nous avons émis l’hypothèse qu’une combinatoire de partenaires protéiques différents permet de moduler l’action d’Otx2 en sélectionnant des cibles génomiques distinctes. Pour identifier cette combinatoire in vivo et la corréler aux fonctions exercées par Otx2, nous avons créé une lignée de souris exprimant une protéine de fusion Otx2-TAP-tag à un niveau physiologique. Cet outil permettra la purification des complexes protéiques Otx2 in vivo et leur identification par analyse protéomique. / In the present work, we study the Otx2 transcription factor as a model to understand how developmental genes achieve multiple functions throughout time. Otx2 is first implied in gastrulation, and then participates to the development of the eye, the olfactory system, the pineal gland, the thalamus and the craniofacial region. Otx2 is expressed in two distinct tissues: retinal pigmented epithelium (RPE) and neural retina including photoreceptors. Global Otx2 gene ablation leads to exclusive photoreceptor degeneration although most of the affected genes are RPE specific. These elements suggest a non-cell-autonomous mechanism, confirmed by RPE restricted gain and loss of function. To understand Otx2 functions in the neural retina and in the RPE, a large scale study of its genomic targets has been yielded. Genome occupancy profiles in RPE and neural retina suggest different Otx2 functions. In the neural retina, Otx2 genome occupancy profile is very close to the one of its paralogue Crx, indicating functional redundancy between both transcription factors. We hypothesized that a different combination of protein partners allows modulating Otx2 action by selecting distinct target genes. To identify Otx2 combinatory in vivo and correlate it to Otx2 functions, we produced a mouse line expressing an Otx2-TAP-tag fusion protein at physiological level. This tool will allow purification of Otx2 protein complexes in vivo and their identification by proteomic analysis.

Aberrant androgen receptor (AR) activity plays a critical role in the development and progression of both early-staged organ-confined and late-staged metastatic human prostate cancer. Recent large-scale genomic sequencing studies showed that ~50% of organ-confined prostate cancer patients have genetic rearrangements that placed the ETS transcription factors (e.g. ERG, ETV1) under the control of androgen-regulated gene promoters such as TMPRSS2. This results in the upregulation of the ETS transcription factors’ expressions in the presence of androgens. The aberrant overexpression of the ETS transcription factors are shown to induce the expression of genes that promote the cellular motility and invasive potential of prostate-tumor cells. Moreover, the improved therapeutic outcome of the second-generation anti-androgen therapies (e.g. abiraterone and enzalutamide) are encouraging, and prove that aberrant AR activity still drives the progression of metastatic prostate cancer. Although these treatments are initially effective, these cancer cells eventually develop resistance to these AR-targeted therapies termed castration-resistant prostate cancer (CRPC). Since the molecular steps involved in AR activation is still not clearly defined, it is critical to define the interactions required for AR activation prostate cancer cells, which will provide a framework for establishing more effective treatments to inhibit aberrant AR activity in human prostate cancer cells. Here, I developed a cellular system to isolate ligand-dependent interactions of AR in prostate-tumor cells. A siRNA luciferase screen was also developed and identified novel modulators of AR-mediated transcription selected from the proteomic dataset. Further biochemical studies showed that AR is associated with the Golgi membrane in a ligand-sensitive manner. And that the nuclear localization of ARA160, an AR coactivator, is regulated by the COPI retrograde trafficking machinery. Collectively, these results support the use of this cellular system to decipher the known AR-interacting proteins and novel components involved in AR signaling in prostate-tumor cells. I next investigated the androgen-sensitive AR transcriptional complexes and androgen-sensitive microsomes isolated from LNCaP prostate-tumor cells. Both studies yielded results that would further strengthen the diverse AR actions mediated within the cell. These results further support the notion that there is significant crosstalk amongst different cell surface receptor signaling pathways with AR. An extension of the androgen-sensitive microsome findings also led us to study the androgen-sensitive G-protein coupled receptor, CXCR7. I showed that androgens regulate the expressions of CXCR7 and CXCR4 and in turn modulated CXCL12-mediated motility in prostate tumor cells. Lastly, biochemical strategies were developed to detect differences in glycoprotein expression of frozen prostate cancer tissues isolated from human patients. I showed that the workflow successfully solubilized and isolated N- and O-linked glycoproteins from the frozen tissue samples and can be analyzed by quantitative mass spectrometry. This workflow would thus facilitate future biomarker studies. In summary, these data demonstrate the utility of developing methods for the comprehensive mapping of AR-mediated signaling in prostate cancer cells, and thus provide novel target candidates for the therapeutic treatment of metastatic or CRPC.

(IN ENGLISH) Structural mass spectrometry (MS) is an evolving field of structural biology introducing novel techniques for the characterization of biomolecules. Although MS-based techniques only can provide "low-resolution" information compared to standard high-resolution techniques representing by X-ray crystallography, cryo-electron microscopy or nuclear magnetic resonance, its uniqueness lies in the ability to easily obtain structural information about various biomolecules in their native or native-like environment. By employing various approaches, from protein covalent labelling through chemical cross- linking to ion mobility, structural MS provides insight into the structure and dynamics of proteins and their complexes over a broad timescale. This thesis is dedicated to the development of novel structural MS approaches based on pulse covalent labelling and chemical cross-linking. Employing the developed quench-flow microfluidics apparatus, we performed footprinting experiments on proteins and protein complexes in timescale from a few microseconds to single seconds. Specifically, fast photochemical oxidation of proteins (FPOP) and novel fast fluoro alkylation of proteins (FFAP) techniques were utilized to track structural changes of myoglobin upon release of the prosthetic heme group....