Advanced solid-state multi-stage biomolecule separations

The development of tools/technologies underpins advances in our understanding of biological systems. A key approach is proteomics, and with it the use of mass spectrometry Swansea University has an outstanding reputation in mass spectrometry. The EPSRC national centre has been at Swansea for over 21 years, and recently moved to the School of Medicine with a more life-science facing outlook. Advances in proteomic technologies have occurred in recent years with e.g. differentially-labelled proteins analysed through 2D gel electrophoresis, and sophisticated liquid chromatography such as MudPit. Both have been coupled with mass spectrometry developments, for 'bottom up' 'middle down' and 'top down' approaches, for biomolecule analysis that continues to offer improvements in accuracy/sensitivity for protein identification/structure. Despite these advances bottlenecks still occur at the sample preparation stage. Disposable pre-LC column based technologies have proved important in proteomics, yet are largely limited to functionalised agarose/sepharose 'beads' for mainstream application. Such systems are limited by the requirement for specialist packing, loss of free 'beads' and therefore surface availability, and sample loss through 'dead space' in columns. To attain the necessary throughput, proteomics continues to advance, requires a step change in early process separation technologies. We propose to develop a solution through the rational application of functionalised solid state, agarose free, separations. Base technology, BioVyon, has been developed Porvair (CASE partner). This early stage technology has not been developed or applied for the proteomics field. This project will develop a high-throughput serial-filtration approach for the rapid sample preparation necessary in advanced proteomics. Columns assembled for differential multi-stage biomolecule separation will be developed for separation of complex protein samples. Simulation of online separation strategies will enable optimal online column design, and inform future functionalisation strategies. The flexibility of this solid-state approach has the potential to impact significantly on high throughput proteomics. Objectives: 1. Develop and evaluate (by protein blotting and mass spectrometry) methodologies for peptide capture using IgG capture from defined peptide mixtures. This will encompass protocol development for proteomics solutions, and will inform design parameters (flow rates, binding times, sample recovery and purity) for subsequent serial-purification. 2. Development of method simulation using Drylab software, and other relevant packages, to evaluate potential serial-purification technology design. Inputs of experimental data from 1, 4, and 5 will continually inform model development. 3. Develop co-sinter functionalised silica e.g. C18 or C8 into HDPE resins by immobilising sorbent particles in an HDPE matrix. SPE sorbent bed weight can be reduced significantly without the channelling problems and the dead volume associated with the inert support frits used for traditional SPE columns. 4. Combine IgG-functionalised BioVyon 'filters' with standard hydrophobic sorbent 'filters' to provide a single-pass solution for protein purification. The co-sintered C18/C8 frit would form part of a simplified 'filter-stacking' system as a precursor to multistack approach. 5. Develop and evaluate simulation-informed options for 'mix and match' column inserts in order to explore the maximum potential as well as limitations of solid-state matrices. Existing purification chemistries as well as novel approaches such as DNA aptamers will be explored. 6. Provision for a 4 month write up period, and 2 month 'transition' peiod towards the end of the research plan. At the end of the studentship we aim to have a very detailed understanding of biological separations, there application in proteomics studies, and the use of mass spectrometry for biomolecular analysis.