TOWARDS AN AUTOMATED APPROACH FOR PROTEIN IDENTIFICATION IN PROTEOME PROJECTS

The development of automated, high throughput technologies for the rap id identification of proteins is essential for large-scale proteome pr ojects. While a degree of automation already exists in some stages of the protein identification process, such as automated acquisition of m atrix assisted laser desorption ionisation - time of flight (MALDI-TOF ) mass spectra, efficient interfaces between different stages are stil l lacking. We report the development of a highly automated, integrated system for large scale identification of proteins separated by two-di mensional gel electrophoresis (2-DE), based on peptide mass fingerprin ting. A prototype robotic system was used to image and excise 288 prot ein spots from an amido black stained polyvinylidene difluoride (PVDF) blot. Protein samples were enzymatically digested with a commercial a utomated liquid handling system. MALDI-TOF mass spectrometry was used to acquire mass spectra automatically, and the data analysed with nove l automated peptide mass fingerprinting database interrogation softwar e. Using this highly automated system, we were able to identify 95 pro teins on the basis of peptide mass fingerprinting, isoelectric point a nd molecular weight, in a period of less than ten working days. Advant ages, problems, and future developments in robotic excision systems, l iquid handling, and automated database interrogation software are disc ussed.