Optimised gel electrophoresis to identify biomarkers of cardiac disease

Objectives: Proteomic technology allows us to examine the overall changes in proteinexpression in the diseased heart so we can generate new diagnostic and therapeuticmarkers. Proteomic studies use mono-dimensional gel electrophoresis (1-DE) and twodimensionalgel electrophoresis (2-DE) with immobilized pH gradients to separateproteins from a sample and combine it with mass spectrometry technologies to identifyproteins. In addition, 2-DE provides a map of intact proteins, which reflects changes inthe level of protein expression, isoforms, or post-translational modifications. We aimedto develop a reproducible and reliable method using gel electrophoresis to identify newcardiac protein biomarkers of 80-400kD for diagnosis cardiac disease.Materials and Methods: we optimised protein solubilisation to collect as many proteinsas possible, after we have optimised voltage migration, sample dilution and gel porosityto have clear and well-resolved bands. We also optimised rehydration type to have amaximum of spots in gel.Results: We have shown that the depletion of high-abundant proteins improved thevisualization of less abundant proteins present in human plasma; Secondly the precipitationwith acetone minimizes dramatically the number of low abundant proteins,thirdly a depleted and undiluted plasma presents more bands than undepleted and/ordiluted plasma; Finally, we also found that using optimized isoelectric focusing electrophoresismigration phase as compared to standard migration phase, improved theseparation of proteins and improved resolution of two-dimensional gel electrophoresis.Conclusions: We discovered new biomarkers of high sensitivity and specificity that canbe used in new diagnostic and therapeutic markers in cardiac disease.Key words: Immunisation OPD–Exclusive breast feeding–Colostrum–Demand feeding