The aim of this proposal is to develop unique facilities for proteome analysis and to make them available to the European Community: this will be achieved through three objectives.
The first objective is to develop a unique two dimensional (2D) gel electrophoreis system. This will be simplified for general use and developed further (e.g. narrow range immobilized pH gradients and a new protein visualization system). Yeast will be used as a model system, using strains where individual genes either have been deleted or are over-expressed, to identify the role of each gene in cellular physiology.
The second objective is to be able to interpret the changes with reference to known proteins. More than 1000 proteins will be positively identified and the post translational modifications of up to 250 proteins will be precisely described by MALDI nano-electrospray or ion trap mass spectrometry (MS).
The third objective is to encourage others to adopt the standardised gel system so that they can interpret their own results using any or all of the data in the database
The uniqueness of the 2D gel system lies in its capability to produce highly -comparable, high resolution 2D gel patterns of yeast proteins in a variety of laboratories. This breakthrough opens a number of exciting possibilities. Because the images are so similar, all the image data can be built into a single image-based database This will be the first time ever that different laboratories will be able to contribute 2D gel data to a common database. It will also permit and encourage the participation of additional labs: each lab that establishes this gel system will thus be able to interpret their own data with the full weight of the entire database. To make this possible we will develop new user-friendly software.
Studies of gene deletion or over-expression mutants would however be valueless without information on the proteins' identity and characteristics. Developments in MS now allow the rapid positive identification of many yeast proteins recovered from gels. In addition, state of the art technologies permit determination of the position and type of post-translational modification
- key data when one wishes to understand he functional regulation of proteins. Many new methodologies will be implemented including robotics to accelerate sample handling for: protein recovery from the gel, enzyme digestion and target preparation and 'on line' advanced software will accelerate mass spectra interpretation. All detectable proteins will be characterised by a variety of physiological characteristics including their cellular localization and turnover.
Finally, extensive measures (including manuals, samples, electronic access to the database, mixed media discussions and training courses) will be taken to assist others in establishing the standardised analysis system used here so that they can analyse and interpret the function of genes in their own projects.
Thus, the ultimate objective of this proposal is to develop new possibility for analysis so that Europe can remain at the leading edge of this rapidly expanding field. Applied to the yeast proteome, these facilities would allow the realization of the true value of the sequencing effort.
Funding SchemeCSC - Cost-sharing contracts
751 82 Uppsala
CB4 4GS Cambridge
5230 Odense M
5230 Odense M
413 90 Göteborg