Award details

Field gradient focusing for proteomic analyses

Reference	BB/C006771/1
Principal Investigator / Supervisor	Dr Richard Ansell
Co-Investigators / Co-Supervisors	Professor John B C Findlay, Dr Jeffrey Keen
Institution	University of Leeds
Department	Sch of Chemistry
Funding type	Research
Value (£)	245,468
Status	Completed
Type	Research Grant
Start date	08/06/2005
End date	07/06/2008
Duration	36 months

Abstract

Field gradient focusing (FGF) is a novel electrophoretic technique where hydrodynamic flow through channel is opposed by a curved electric field, such that proteins loaded in the channel focus at a point determined by their electorphoretic mobilities. Unlike isoelectric focusing, focusing occurs with proteins in the charged state, not at their pl, such that problems with precipitation are avoided. Focusing also occurs significantly faster. In the proposed project, we plan to develop an FGF instrument, building on the main applicant¿s experience with a prototype but improving particularly on the electrode control system, and on methods of visualisation of proteins in the separation channel, and developing methods for the selective elution of focused bands for further analysis. We will then apply the instrument to the analysis of plasma proteins and membrane proteins. We will improve on the current design by building a twenty-variable electrode system, investigate the use of different packing materials in the separation channel and different chamber designs (in particular, miniaturisation). Visualisation of non-coloured proteins will be investigated via moderation of fluorescence from a fluorescent packing material, UV transmittance and UV reflectance, detected via a UV CCD. Chamber designs incorporating a mid-point exit will be compared with a simple design with only one outlet, at the bottom, for the controlled, one-fraction-at-a-time elution of model protein mixtures. With the plasma proteins, eluted fractions will be subjected to digestion followed by peptide mass fingerprinting using MALDI-MS and/or QToF-MS/MS to identify the protein species. This will give us a good indication of the degree of purity and hence of the separation achieved. 2D-PAGE analysis of apparently single bands focused under non-denaturing conditions should reveal minor components which interact with or are simply obscured by the major proteins. Membrane samples will be studied using detergents and solubilising agents in the channel running buffer. Again, off-line digestion and MS will be investigated. Selective visualisation of the GFP-tagged GPCRs in the separation channel should be possible with a near UV source and visible camera. As with the plasma proteins, eluted fractions will be further analysed to obtain information about the interacting proteins. Flow from the FGF instrument directly into a second separation dimension, RP-LC, and into the ES interface of a quadrupole MS will be investigated. On-line RPLC and MS of eluted bands will be investigated.

Summary

unavailable

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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