Year of Publication
Arts and Sciences
Bert C. Lynn
The development of a highly sensitive ruthenium-based fluorescent staining solution isdescribed in this dissertation. The in-house synthesized ruthenium complex (RuMS)containing both sulfonated and non-sulfonated ligand has detection limit of 1 ng ofprotein that is better than colloidal coomassie, silver and ruthenium complex containingall sulfonated ligands (RuBPS). RuMS stain has 100-fold dynamic range and does notinterfere with subsequent mass spectral identification of proteins. The capability of inhousesynthesis of the staining solution makes it a viable cost-effective alternative to theexpensive commercially available fluorescent stain, Sypro Ruby. The low detection limit,broad linear dynamic range and compatibility with mass spectrometry, make thedevelopment of this stain a worthwhile pursuit. The staining solution was utilized insubsequent applications of two-dimensional gel electrophoresis (2-DE) technology.Proteomics methodology utilizing 2-DE and mass spectrometry was applied toinvestigate the effect of malathion on the proteome of human neuroblastoma cells.Results indicated that out of 122 proteins that were identified from the neuroblastomaproteome, sixteen proteins were down-regulated while five proteins were significantlyup-regulated after treatment with malathion. Significant down-regulation of calciummodulators like calmodulin and calgizarrin and other key chaperones makes themalathion-treated cells highly prone to oxidative stress. With increased awareness inpesticide related adverse effects, identification of altered proteins in malathion-treatedhuman neuroblastoma cells is a critical finding.Proteomics is a major area of research in the identification of biomarkers for diseases. Anovel immunoprecipitation method developed in this work allowed for successfulisolation and identification of albumin-interactome in cerebrospinal fluid (CSF) that isusually under-represented in standard CSF analysis using 2-DE. A key finding is thedifferential expression of various isoforms of proteins in CSF albumin-interactome fromAlzheimer's disease (AD) subjects. The data implicate the acidic isoform ofprostaglandin D2 synthase (PGDS2) as a potential biomarker for AD. An understandingof the differential expression of these protein isoforms in AD will provide insight into theetiology of the disease and this can have far-reaching implication on drug developmentleading to the cure or even preventation of the disease.
Rajagopal, Meena Uma, "METHODS DEVELOPMENT AND APPLICATION OF TWO-DIMENSIONAL GEL ELECTROPHORESIS AND MASS SPECTROMETRY IN PROTEOMICS" (2006). University of Kentucky Doctoral Dissertations. 292.