Djuro Josic is a professor of medicine (research) at Brown and director of the Proteomics Core in the Center of Biomedical Research Excellence Center for Cancer Research Development (COBRE CCRD) at the Rhode Island Hospital. One of main goals of our Core is to engender young investigators with broad scientific capabilities, thereby optimizing their productivity and funding opportunities. To this end, the facility works closely with investigators to ensure experimental success by offering consulting, experimental advice, and help with reagent and protocol development.
In response to facility user needs, we have developed the technology for isolation of highly pure organelles from cell homogenizates by use of monoclonal antibodies specific for particular organellar proteins. These antibodies can be immobilized onto magnetic beads or highly porous monolithic supports. Using this new system, we can fractionate whole cells and isolate highly pure organelles, with the final goal to characterize cellular proteins and enrich low abundance proteins for further separation and identification by use of mass spectrometry (MS).
In cooperation with the Molecular Pathology Core also belonging to the COBRE CCRD Program (Murray Resnick and Patricia Meitner) and together with a COBRE CCRD Investigator, Steven Moss, we established a new method for the identification of proteins after extraction from formalin-fixed tissue samples. This method will make possible retrospective analyses and direct comparison of normal and malignant samples from the Tissue Bank in the Molecular Pathology Core.
In cooperation with A. Grollman from the State University of New York at Stony Brook and the University of Zagreb, Croatia, we are starting proteomic analyses of urine and serum from sensitive and non-sensitive animals after treatment with aristolochic acid. This substance is suspected of causing chronic kidney failure in men, known as Balkan Endemic Nephritis (BEN), that can result in the development of urothelial cancer. This mouse model is further useful to better understand the kidney failure that is caused by this environmental agent and sometimes ends with kidney or urothelial cancer.
In cooperation with Werner Reutter, Professor of Medicine at the Humboldt University in Berlin, we are developing methods for the isolation of plasma membranes from rat liver and hepatocarcinoma Morris hepatomas 7777 with minimal contamination from other organelles by use of monolithic supports and magnetic beads (see above). Additionally, methods for identification of hydrophobic, integral membrane proteins by LC/MS-MS were developed. These proteins are difficult to identify because of very low yield if routine protocols for tryptic digestion and LC separation of hydrophobic peptides and glycopeptides are applied.
We also established a collaboration with G. Silverberg, professor of neurology at Brown University in order to identify biomarkers for Alzheimer's disease. Samples of treated and non-treated patients were compared by SELDI-TOF mass spectrometry. After statistical analysis of mass spectra of cerebrospinal fluids, possible biomarkers able to distinguish patients that respond to the treatment from non-responding patients were identified. Identification of biomarkers is the first step for fast and simple diagnosis of Alzheimer's disease and for simple and rapid follow up of disease treatment.