Award details

Multidimensional chromatography for high throughput post-genomic pharmaceutical science

Reference	BB/C511356/1
Principal Investigator / Supervisor	Professor William Griffiths
Co-Investigators / Co-Supervisors	Professor Laurence Patterson, Professor Frances Stephenson
Institution	University College London
Department	Dept of Pharmaceutical and Biological Ch
Funding type	Research
Value (£)	45,980
Status	Completed
Type	Research Grant
Start date	11/07/2005
End date	10/07/2006
Duration	12 months

Abstract

Today, the related disciplines of proteomics and metabolomics are progressing from providing a global identification of the biomolecular make-up of cells and tissues, to providing a quantitative measure of these biomolecules. Further more, the qualitative and quantitative variation of the biomolecular make-up of cells and tissues in different functional states is now being addressed. However, the dynamic nature of biology adds the dimension of time to such studies making high-throughput technologies desirable. At the School of Pharmacy we are following a functional proteomic approach to the qualitative and quantitative analysis of cytochrome P450 (CYP), and other proteins of pharmaceutical importance, from different tissues in different functional states. We have identified a number of limitations to the conventional proteomic approaches to protein identification, which compromise the biological significance of data obtained. Specifically, most proteomic approaches follow bottom-up methodologies, where by a protein is identified by observation of a just a few marker proteolytic fragments. Much information on primary protein structure is thereby lost. Further more the functional activity of a protein is not generally considered. The polymorphic nature of CYPs, and other drug metabolising enzymes, enhances the importance of maximising primary structural information for both qualitative and quantitative studies. In the current proposal we plan to extend our studies to include both bottom-up and top-down proteomic methodologies to maximise primary structure information, and also add a functional activity element to our studies. We will revise our analytical strategy by introducing a multidimensional liquid chromatography (LC) element to our work-flow. We will follow the following basic strategy: Following a sub-cellular fractionation step, or affinity pull-down; proteins will be separated by on-line multidimensional LC involving ion-exchange and reversed-phase (RP) steps. The eluting protein fractions will be split so as to allow on-line mass analysis of the intact proteins and fraction collection. The protein fractions will then be annotated with their contents molecular mass, and subsequently digested with proteolytic enzymes eg. trypsin. The resulting peptides will then be separated in a second two-dimensional (2D) chromatography procedure, consisting of cation-exchange and RP dimensions. This revised work flow is expected to show the following advantages: We have shown in a preliminary study that the application of 2D chromatography to tryptic digests of proteins significantly increases the observed sequence coverage. This will be particularly important for the identification of polymorphic and posttranslationally modified proteins. Further, the additional peak capacity provided by the two dimensions of chromatography increases both the sensitivity and the dynamic range of the analysis. Once a protein has been identified via this bottom-up methodology, the data will be correlated with the measured protein mass. Discrepancies between sequence mass and measured mass will indicate a variation in primary structure. At which point, the tryptic digest data will be researched using a specially constructed error-tolerant data-base search to allow for posttranslational modifications (PTMs) and single nucleotide polymorphisms (SNPs). It is expected that the work-flow using two steps of 2D chromatography will allow the high throughput characterisation and quantification of functional, posttranslationally modified and polymorphic proteins.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Research Equipment Initiative 2004 (RE4) [2004]
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search