Proteomic analyses of bioactive molecules in health and disease

Abstract: After the mapping of genomes and knowing the blueprints of the possible gene products, focus is shifting from genomics to proteomics. Proteome analyses can be used for diagnosis, prognosis and monitoring of diseases and for characterization of bioactive compounds and their mechanisms of action. Post-translational modifications, as for example phosphorylations, are important for activity of many bioactive molecules. In paper I, a novel compact disc (CD) based microfluidic method for detection and enrichment of phosphopeptides was tested. Peptides were on-CD processed using immobilized metal affinity chromatography (IMAC), followed by on-CD MALDI mass spectrometry. In about 200 analyses, with a BSA tryptic peptide background, successful detection of phosphopeptides after IMAC enrichment was achieved in 94% of the cases. Applied to the ligand-binding domain of the human mineralocorticoid receptor the method identified two novel phosphorylation sites. 2D-gel electrophoresis (2-DE) is a well established technique in proteomics. Although with limitations, proteome analysis can supply us with important information on biological materials. Comparisons of patterns in 2-DE obtained from normal versus specific disease states can be used to trace marker proteins. In paper II, a proteome analysis of Vernix caseosa by 2-DE and mass spectrometry gave us insight into the role of vernix; we identified 41 proteins of which 25 were novel to vernix and nearly 40% turned out to have a role in innate immunity. Continuing with 2D-gel separations whole cell extracts from isolated drug-treated and from native pancreatic rat islets were separated by 2-DE in paper III. The silver stained gels were scanned, processed by the image analysis tool PDQuest and 22 differentially expressed proteins were identified by mass spectrometry, 15 up-regulated and 7 down-regulated. In paper IV, we directly studied molecular interactions of the insulin releasing imidazoline compound (BL11282) from paper III using SPR technology and affinity purification with magnetic beads. We identified two proteins binding to the drug, elongation factor 1 alpha and glucose regulated protein 94. In patients with chronic kidney disease (CKD), peptides and proteins circulate in elevated concentrations. It is estimated that more than 106 different protein components are circulating in the plasma, but a few abundant proteins contribute to 99% of the protein mass. A multiple affinity removal system was employed in paper V, followed by a workflow including HPLC and SDS-PAGE separation and subsequent protein identification by mass spectrometry. We identified 29 proteins present at altered levels in CKD plasma compared to the amounts in plasma from healthy individuals. In paper VI, we again study molecular interactions in relation to diabetes. An oxidative cross linking method was optimized and applied to the proinsulin C-peptide. An interaction partner of biological significance (protein tyrosine phosphatase 1B) was identified, and the concept of cross-linking affinity purification, CLAP, was introduced. Proteome analyses include a wide variety of methods. The selection of methods is of great importance for the outcome of proteomic studies, and the need for method development is not expected to cease. In this thesis, a selection of both well established and novel proteomic methods was used to study bioactive molecules.