This project addresses the application of high resolution and high sensitivity mass spectrometry to characterize the protein components in rodent scent marks. Recent research in rodent semiochemistry, in substantial part from the academic applicants laboratories, has revealed a depth and complexity to rodent chemical communication previously unanticipated. We are building a detailed picture of the receptor repertoire for these signals, and of the higher level processing that collates these signals into behavioural responses, but our understanding of the molecular composition of the scent marks is some way behind. Chemical communication is capable of conveying an incredibly subtle stream of information, especially between conspecifics. Scent marks, predominantly urinary, contain an astonishing array of information that transmits variable indicators of the state of the scent owner (e.g. health status, pregnancy, recent food ingested and time since deposition). This status information is primarily associated with proteins (lipocalins and ESPs) that provide information on genetically invariant parameters such as sex and individual identity. In this project, the student will bring to bear advanced mass spectrometric methodologies in the characterization of these proteins. The samples will be recovered from wild-caught rodents, and are sometimes vanishingly small (such as in tear secretions). The challenges in the study of these scent mark proteins are two fold. First, there is a pressing need for accurate quantification of the proteins in the scent mark. Second, it is clear that the highly polymorphic gene cluster that encodes these proteins is genetically unstable, leading to each wild animal being able to express a unique pattern of these proteins. Thus the challenges are both quantitative and qualitative. The student will address these challenges in collaboration with Waters, using a combination of intact mass profiling, making use of the high resolution QToF instruments and new algorithmic approaches to spectral deconvolution developed at Waters, label-free quantification using the Waters-developed MS^E analytical workflow, coupled with Hi3 peptide quantification, and through the use of selected reaction monitoring, using an artificial QconCAT concatenated standard peptide assembly (technology invented and patented by the academic partner). Finally, discovery of new polymorphic variants will be based on intact mass survey, followed by electron transfer dissociation (ETD, using a new front-end source designed at Waters but not yet widely available) to isolate and characterise the amino acid sequences of the variant proteins. The plan of the programme will follow the outline below, although we expect that from year 2 onwards, the student will take some responsibility for the direction of the research. Year 1: Induction, Design of QconCAT proteins, expression and validation, experience of wild rodent sample collection and diversity. Training in intact mass profiling, bioinformatics tools and peptide level label-fre and label-mediated quantification. Year 2: Development of ion mobility methodologies to improve resolution of complex mixtures of isoforms, and the use of gas phase cross-sectional area to assess the conformational stability and consequent degree of protonation on electrospray ionsation behaviour. Expression of recombinant lipocalins for model studies, appropriately engineered to alter electrostatic potential for charge state manipulation. Instruction on ETD fragmentation. Year 3: Application of ETD to discover amino acid sequence variation in new isoform variants, leading to quantification by surrogate peptides. Includes an exploration of the effect of sequence variation on ESI signal intensity and charge state for quantification. Year 4: Completion of thesis and papers, exploration of new areas of investigation.
We propose to take a proteomics approach to the study of protein secretion and turnover in two yeasts: the microbial cell factory, Pichia pastoris, and the model eukaryote, Saccharomyces cerevisiae. We shall use a proteomics based approach to look, in both species, at the dynamics of transport of a heterologous protein from the site of its synthesis to the cell exterior. In both hosts we shall use recombinant human lysozyme (HuLy) as the test object. Previous work has demonstrated, using a series of lysozyme mutants, that the degree of unfolding of HuLY is a major factor in determining its secreted yield (1). Highly unfolded variants show poor secretion yield and trigger the unfolded protein response (UPR). We shall use a global proteomics approach to determine where in the secretion pathway from the ER to the cell exterior lysozyme and its variants accumulate. Highly unfolded proteins are known to induce both ER stress and the unfolded protein response (UPR). Our rationale for taking a global approach to dissect the secretion pathways of heterologous proteins in yeasts, is based on the need to determine not only the subcellular locations at which such proteins accumulate, but also their binding partners within each specific location. Standard methodologies to capture protein complexes using immunopreciptation of a bait do not yield information about compartmentalisation of intermediates and the dynamic nature of binding partners within compartments. We will thus make use of state-of-the-art technologies developed in the Lilley laboratory which allow accurate assignment of proteins to subcellular locations using distribution patterns of subcellular compartments on density gradients as determined by quantitation proteomics methods coupled with sophisticated statistical tools (2). In this project we will work with Jim Langridge who is Director of Proteomics at Waters to further develop this method employing up-to-date label-free proteomics methodologies to determine the distribution of thousands of proteins simultaneously. This label-free approach has been pioneered by Waters and has many advantages over the methods that the Lilley lab. has used to date, namely isobaric stable isotope in vitro labels. Recent work by the Lilley lab. has shown that these labels, such as iTRAQ, have significant problems regarding both their precision and accuracy (3). Robust label-free approaches have been shown not to suffer from the same shortcomings as the iTRAQ tags (4,5) and their use in determining the distribution patterns of organelle proteins within density gradients are more likely to lead to accurate measurement of such patterns and thus better resolution of the patterns associated with different sub cellular structures. Moreover, the label-free method to be employed, MSE, also estimates the absolute amount of proteins within different fractions, enabling measurement of stoichiometeries of proteins in complexes, as absolute distributions of protein species in terms of molecules of protein per compartment. Having further developed label free quantitative proteomics approaches to determine methods accurate subcellular locations of proteins and their binding partners, we will focus on examining the compartmentalisation of the recombinant protein. We will carry out global analysis of its association with other proteins including the unfolded-protein chaperone, Kar2p (a BiP ortholog) and the proteasome. We shall be particularly interested in the amyloidogenic version of HuLy (I156T) and will validate our results using this variant by expressing the Alzheimer's protein Abeta, both in its native form and as Abeta42 -GFP fusions. 1. Kumita, JR et al (2006) FEBS J273(4):711-20 2. Dunkley, T et al, (2006)Proc. Natl. Acad. Sci 103(17):6518-23 3. Karp, NA et al (2010) Mol. Cell Prot. in press 4. Silva, JC et al (2005), Anal Chem. 1;77(7):2187-200 5. Stapel, M et al (2010) Sci Signal.2;3(111)
Enzyme catalysis is being industrialised at a phenomenal rate, offering routes to chemical transformations that avoid expensive heavy metal catalysts, high temperatures and pressures, and providing impressive enantio-, regio- and chemo-selectivities. In short, biocatalysts are a cornerstone of the bioeconomy: they are required individually, or as cascades, in live cells or cell-free preparations to manufacture every day chemicals, materials, healthcare products, fuels and pharmaceuticals; and they are integral to many diagnostic and industrial sensing applications. They are central components of technologies underpinning the circular economy and offer engineering biology routes to realising global challenges, including net zero, clean growth and the bioeconomy. An ability to exploit and tailor biocatalyst activities both rapidly and predictably is essential to realising the contemporary global challenges and the UK Government's Innovation Strategy. Despite their central importance, the vast majority of natural and engineered enzymes are thermally-activated. This dependence on thermally-activated catalysis: i) limits biocatalysis to those reaction types found naturally in biology; ii) places a high dependence on expensive and unstable cofactors / coenzymes; and iii) places a sizeable demand on the provision of energy source (biochemical / artificial reductants), 'bioreactor' designs (e.g. within cell-free formats, nanoscale devices or microbial cell factories); and iv) restricts approaches to regulating biocatalyst / bioprocess activity. The use of light to drive enzyme catalysis would bypass many of these hurdles. However, with only three known exceptions, nature does not make use of enzymatic photocatalysis. Therefore, biology cannot access a broad range of 'difficult-to-achieve' reactions that would be transformational in catalysis science, and applications of these reactions in the modern world. Light is freely available and non-invasive, yet the photochemical versatility of natural cofactors such as flavin is seldom used by enzymes. Therefore, securing generalised routes to predictive photobiocatalysis design is a fundamental biological challenge. If successful, identifying generalised routes to the engineering and design of photobiocatalysts would be transformative for catalysis science in the emerging bioeconomy. This project will address this urgent need by using the natural photochemistry of flavin to make possible photocatalysis by any flavin-containing protein. This programme (termed GENPENZ) is positioned at the frontier of biological photocatalysis and enzyme design and engineering. It will generalise the concept of photo-biocatalyst design and engineering using existing (top down) and man-made (bottom up) protein scaffolds to biologically encode new photo-biocatalysts with wide reaction scope, or to assemble de novo protein frameworks from synthetic peptides. It will unite time-resolved 1D / 2D spectroscopy in the visible / infra-red spectral regions, across 12 decades of time (fs - s), with emerging capabilities in photo mass spectrometry (ion mobility; hydrogen-deuterium exchange), EPR spectroscopy, and photo-biocatalyst design engineering. High-level computational chemistry will underpin all protein-design/engineering work, spectroscopy, and structure elucidation. GENPENZ is based on breakthroughs in discovery science relating to mechanisms of enzyme photocatalysis. Realisation of a generalised platform for photo-biocatalyst design will open up new high-energy reaction pathways, enrich catalysis outcomes, and sidestep many of the scientific / economic constraints of working with thermally-activated biocatalysis in the emerging bioeconomy.
The development of 'soft ionisation' techniques have positioned mass spectrometry as the central go-to technique for proteomic investigations. Electrospray ionisation (ESI)-MS is used extensively in this post-genomic era to determine the primary structure of proteins and has really become the essential tool in so called 'bottom-up' proteomic analysis. Extensive effort and resource has gone into mass spectrometry based proteomics, the majority of which relies on so called 'bottom-up' characterization where proteins are enzymatically cleaved into peptides for MS analysis followed by database correlation to identify (and quantify) the proteins under study. This approach has many analytical advantages; critically it is high throughput and sensitive and clearly has succeeded in many studies however it has some drawbacks. Perhaps the most obvious is that "bottom up" approaches cannot provide direct information on the active fold and interactions of the proteins being analysed, this limits the functional data that could be obtained from these studies. The analytical advantages of mass spectrometry also apply to its use to examine intact proteins and complexes and there is an emerging research field that identifies proteins this way - so called "top-down" methodologies where proteins are sequenced in the mass spectrometer. Most top down approaches destroy the functional form of the protein prior to sequencing, to enable higher throughput and to facilitate more productive analysis from higher charged parent ions. This is not quite the route we will take, rather we will probe intact proteins and complexes , with careful use of nano-electrospray ionisation (nESI) to retain solution structures. This proposal will develop so called "top down" methods to examine conformations and dynamics of proteins and protein complexes. The research program will enhance the gamut of predominantly solution-phase based techniques which evaluate protein structure and interactions. Methodologies will assess conformational stability, and dynamics of proteins both in solution and in a solvent free environment. Funds and research time are requested to construct novel instrumentation with which to measure conformations, and unfolding and refolding dynamics over timescales ranging from microseconds to minutes. The proposed instrument will also be able to photo-dissociate mass and conformer selected ions, and detect the product ions. The program of work following technology development will focus on two areas: 1. Top down structural proteomics; combining photo-dissociation and ion mobility mass spectrometry (IM-MS) to map protein structure and interactions. 2. Performing FRET combined with IM-MS, to determine protein unfolding pathways and the effect of fluorescent makers on protein structure. The equipment will comprise a novel duel ion guide mobility mass spectrometer, wherein IM-MS will be used to determine collision cross sections, and optical methods will interrogate structure and stability via FRET and/or photo dissociation. This combined IM-MS photo activation approach will be termed photo-IM-MS. Ions will be externally generated via ESI and transferred into a customised ion mobility mass spectrometer. Once in the duel drift region of the IM-MS, the drift time of ions (under the influence of a weak electric field) is related to their rotationally averaged collision cross section with the buffer gas. This mobility measurement can be made, or alternatively the ions will be 'stepped' into a parallel stacked ring ion guide drift region (2SRIG), which has a laser beam passing through it as well as optical detection. Ions in this region will interact with light and either be optically detected, or be pushed back into the first mobility cell. On exiting the cell ions will be transferred to a time-of-flight mass spectrometer and thus will be detected as a function of both mass, charge state, and cross section and potentially following optical interaction
Amyloidosis is a pathological condition associated with the self-aggregation of proteins into highly ordered amyloid fibrils in vivo. Despite the importance of these high-profile disorders in today's ageing population and a wealth of on-going research, knowledge of the structural molecular mechanism of amyloid formation remains limited, primarily because of the complexity and heterogeneity within these systems, and rationally designed therapies are rare. Several small molecules have recently been found to modulate (stimulate or inhibit) the rate of fibrillogenesis of various amyloidogenic proteins in vitro but, in many cases, their reaction mechanism remains unknown. We have recently developed novel mass spectrometric methods to separate protein conformers and oligomers in real-time. Mass, kinetic and thermodynamic stability, and shape/cross-sectional area of each component within a heterogeneous assembly reaction can be measured in a single experiment using travelling wave ion mobility spectrometry coupled to mass spectrometry (IMS-MS; engineered by Micromass/Waters). Our data demonstrate the immense power of IMS-MS to resolve transiently populated species during the early stages of amyloidosis in vitro and form the basis for this project (Smith et al., J. Am. Soc. Mass Spectrom., 2007; Smith et al., PNAS, 2010). The aim of this project is to define amyloidogenic protein-ligand binding events in detail to pave the way to the rational design of ligands able to inhibit fibrillogenesis. To achieve this we will: (i) assess changes in the protein population (i.e. ratio of protein conformers, population of oligomers, etc.) caused by ligand presence; (ii) define which species bind the ligand; (iii) compare the conformational properties of the protein monomer and oligomers pre- and post-ligand binding; (iv) monitor the effect of ligand binding on the progress of fibril assembly. The student will initially study the protein-ligand binding characteristics of beta2-microglobulin, an amyloid-forming protein with which Ashcroft and Radford have gained much experience. Specifically, the student will study the rifamycin family of small molecule macrocycles, some of which we have found inhibit amyloid fibril formation whilst others have no effect. Using IMS-MS to analyse the mixture of protein conformers and oligomers, we will assess the changes in protein population resulting from ligand presence and identify ligand-binding species. Protein-ligand binding stability will be assessed from their binding constants and by MS/MS collision induced dissociation. To detect any conformational change on binding, the shape/cross-sectional areas of pre- and post-ligand binding protein species will be measured by IMS-MS. The protein regions involved in ligand binding will also be explored using novel HDX-IMS-MS methodology in collaboration with Micromass/Waters. The MS experiments will be complemented by other biophysical measurements (size exclusion chromatography, analytical ultracentrifugation) and the presence of fibrils in each experiment will be confirmed by electron microscopy. Protein mutants with different fibril-forming propensities which have been engineered by Radford will be compared to identify ligand binding residues. Non-amyloidogenic murine beta2-microglobulin will be used as a control. After establishing robust methods, the project will widen to encompass other proteins associated with amyloid diseases. Using the techniques described, their ligand binding properties will be investigated with known inhibitors. This will include the interactions of alpha-synuclein (Parkinson's disease) with baicalein and other flavanoids; Abeta (Alzhiemer's disease) with catechol derivatives; IAPP (type II diabetes) with resveratol. Thus, a correlation between ligand binding, protein conformation and fibril formation will be assessed which will ultimately pave the way for the rational design and screening of amyloid inhibition ligands.