Grasping the proteome: detergents sub-fractionation of human and microbial cells leads to improvements in differential proteomics

Proteomics is an emerging area of science that attempts to study proteins on a massively parallel scale. It is by essence a multidisciplinary science: physics, chemistry, bioinformatics and mathematics join biochemistry, biology and medicine to solve general life science questions. Scientists worldwide are applying proteomic technology to solve problems which cannot be resolved by traditional methods, particularly in the biochemical field. Methodologically, proteomics is based on highly efficient methods of separation and analysis of proteins in living systems, which need to be continuously improved in order to achieve and maintain high resolution standards. Proteomics is considered a key technology in many biomedical sectors such as molecular medicine, drug discovery, clinical diagnostics, as well as microorganisms and plant studies. Shared problem in proteomic analysis is the great complexity of the samples, thus the proteomic technology needs improvements and new contributes to overcome the actual limitations in the samples treatment and recovery, to shorten and ease the recovery protocols, to affirm reproducibility and to effectively remove contaminants, in order to reach the degree of automation and cross-laboratory reproducibility collectively expected by the scientific community. The objectives of this thesis were both the development of protocols for protein recovery in order to achieve higher resolution and reproducibility in 2-DE, and at the direct applications of the set methods to the understanding of two selected and interesting proteomic cases. Concerning microbiological proteomics, we studied the molecular mechanism of acid-resistance in Ga. Hansenii; and concerning the biomedical proteomics, the molecular basis of iron homeostasis in macrophages was studied. The thesis work was performed at the Biochemical Methodology and Proteomics laboratory of Department of Biotechnology of the University of Verona, in collaboration with other laboratories. Regarding the study on macrophages, we collaborated with Dr. Annalisa Castagna and Prof. Domenico Girelli, Unit of Internal Medicine B, Department of Clinical and Experimental Medicine of the University of Verona, with Dr. Ivana De Domenico, Division of Hematology, Department of Medicine, School of Medicine, University of Utah, and Prof. Jerry Kaplan, Department of Pathology, School of Medicine, University of Utah, and with Dr. Anna Maria Timperio and Prof. Lello Zolla, Department of Ambiental Science, University of “La Tuscia”, Viterbo. Regarding the study on Ga. hansenii we collaborated with Dr. Giacomo Zapparoli, Department of Biotechnology of the University of Verona and with Prof. Paolo Giudici, Department of Agricultural and Food Science, University of Modena and Reggio Emilia.