Basic and clinical applications of proteomic research. Our experience.

Abstract

In this thesis we applied proteomic techniques to basic biological topics as well as to clinically relevant issues both in diagnostic and in investigative perspectives. The procedure we employed consisted of electrophoretic separation of the relevant proteins, in-gel-proteolytic fragmentation and finally identification either of precise sites of labelling in irreversible protein PTM by Tgase 2 or of the protein itself in clinically oriented oncologic studies. In protein PTM we have analysed three substrate proteins, troponin, tubulin and mutant dyphtheria toxin. We obtained evidence that TnT was the subunit modified in native troponin and that glutamine 13 is the site of Q-labelling, while the protein is not a K substrate. Data on tubulin and DT are not yet so advanced. Both proteins are however substrates of Tgase 2. Tubulin is better substrate when in the aggregated form and is labelled in the C-terminal region. Moderate crosslinking also occurred. The protein when we focused on is the DT mutant CRM197, which instead undergoes massive crosslinkage forming protein aggregates whose accumulatioon is competed by incorporation of external amines, confirming the occurrence of g-glutamyl-e-lysine isopeptide bonds. In the oncologic studies we used the bi-dimensional electrophoresis and mass spectrometry approach to isolate specific proteins and we identified from breast cancer tissues to detect candidate disease biomarkers for diagnosis and prognosis and proteins involved in Epithelial Mesenchymal Transition in cholangiocarcinoma cell cells. In our four investigations we took particularly of the technical aspects to optimize resolution of the experimental data in both perspectives.