Award details

A high resolution Q-ToF MS for protein modifications protein:DNA interactions and dynamic combinatorial libraries

Reference	REI20520
Principal Investigator / Supervisor	Professor Carol Robinson
Co-Investigators / Co-Supervisors	Professor Sir Shankar Balasubramanian, Dr Jonathan Spencer
Institution	University of Cambridge
Department	Chemistry
Funding type	Research
Value (£)	150,000
Status	Completed
Type	Research Grant
Start date	01/10/2003
End date	31/10/2004
Duration	13 months

Abstract

The main focus of this application is the analysis of proteins, their interactions and modifications. The mass spectrometer will complement existing facilities within the department and will contribute specifically to the following projects: 1. The study of large macromolecular complexes such as ribosomes, spliceosomes and the proteasome. Although it is possible to maintain non covalent protein interactions in these complexes in the mass spectrometer in order to examine the interactions between the different components it is necessary to develop cross linking strategies such that the precise interactions between subunits can be defined. For this approach it is desirable to use a nano LC system capable of interfacing to a Q-tof type instrument with high resolution and MS/MS capabilities. The tandem mass spectrometry capabilities are essential for obtaining sequence information and the high resolution aids in the assignment of the peptide sequence. 2. The research group of Jonathan Spencer is active in understanding the biosynthesis of antibiotics by characterising enzymes involved and monitoring their reactions and modifications. His research group have access to an early LCQ mass spectrometer but resolution and mass range are limited with this instrumentation. 3. Dr Shankar Balasubramanian and his group are interested in DNA sequences that cap human chromosomes, synthesised by the enzyme telomerase. Both the enzyme and the telomeres synthesised by the enzyme are key to biological mechanisms important for the ageing of human cells and the proliferation of cancer cells. The mass spectrometer will be used to identify links between protein and RNA in these macromolecular complexes. His group are also interested in G-quadruplex DNA, non-classical four-stranded DNA conformations adopted by DNA sequences from a number of regions in the human genome. The formation (or disruption) of such structures can influence biological processes that include cell division and the expression of certain genes. Using both protein engineering and organic synthesis the group are exploring these hypotheses at the molecular and cellular biological levels. Access to high resolution electrospray mass spectrometry equipment will aid significantly these projects. 4. The mass spectrometer will be used to characterise metalloporphyrin assemblies. These assemblies are involved in development of nano capsule drug delivery systems based on dynamic combinatorial libraries and are being developed by Prof J K M Sanders and Dr Sijbren Otto. 5. A number of chemical biology projects in the department involve the construction of protein variants. For example the research of Professor Fersht and Dobson, Drs Jane Clarke and Sophie Jackson is directed at understanding the role of critical residues in defining the overall protein fold. Hydrogen exchange combined with electrospray mass spectrometry has proven to be a valuable technique for analysing protein variants. The higher resolution afforded by the proposed instrumentation will aid significantly the progress of these projects.

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 2003 (RE2) [2003]
Funding Scheme	X – not Funded via a specific Funding Scheme

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