Application of chemical proteomics to biomarker discovery in cardiac research

This thesis is primarily focused on (i.) exploring chemical probes to increase sensitivity and specificity for the investigation of low abundant cardiac proteins applicable to both biology and biomarker discovery, and (ii.) exploiting different aspects of mass spectrometry-based proteomics for building a concentration-based cardiac proteome inventory. Chapter 1 is an overview of the applications of proteomics in cardiac diseases, including detailed descriptions of proteomics platforms used to analyze protein expression, function and quantity. It is also illustrated label-based and label-free mass-spectrometry quantitation methods to monitor changes in the proteome. The traditional biomarker discovery approach is described, including animal models and clinical studies. Chapter 2 reviews the specific contribution of mass spectrometry to the understanding of two particular cardiac signaling pathways that evolve around the second messenger cyclic nucleotides cAMP and cGMP. Two major downstream effectors of these cyclic nucleotides are Protein Kinase A and Protein Kinase G. Their relations to scaffold proteins that compartmentalize these kinases are discussed. An overview of mass spectrometry-based studies such as native mass spectrometry, H/D exchange and ion mobility mass spectrometry were described. In chapter 3, a chemical proteomics approach is combined with stable isotope labeling and mass spectrometry to study the specificity of different PKA isoforms for different AKAPs. Three different immobilized cAMP-analogues were used to enrich for PKA from several cell types and rat tissues. Stable isotope labeling was used to quantify the differential enrichment of the PKA-isoforms and thus their interacting AKAPs. Of the twelve AKAPs detected, seven preferentially bound to RII, whereas the remaining five displayed at least dual-specificity with a potential preference for RI. For the first time, the specificity of AKAP14, AKAP2 and AKAP12 could be established. In Chapter 4, cAMP-based chemical proteomics is employed to identify potential changes in concentration and association of PKA in the dilated cardiomyopathy (DCM) affected human heart. Specific enrichment of PKA, PKG, several phosphodiesterases and many AKAPs from both healthy and DCM hearts were quantified in a label-free manner. We confirmed that PKA-R concentrations were lower in DCM affected tissue. Interestingly, the specific interactions of PKA with AKAPs were altered in the diseased heart under DCM conditions. These experiments, demonstrated the powerful combination of chemical proteomics with isotopic labeling as a potential application for cardiac biomarker discovery. Chapter 5 continues with the application of a multiplexed proteomics approach to generate a concentration-based human left ventricle proteome library. By using different separation methods/ proteases/ gas phase fragmentation methods, we identified 3,584 distinct proteins with high confidence which were quantitated using a sophisticated label-free spectral counting method to yield a comprehensive abundance level of human left ventricular proteins. Most currently used CVD-biomarkers, as well as many muscle machinery proteins are present as high abundant proteins in LV. The signaling proteins, protein kinases, phosphatases and small GTPase were studied in the context of their concentrations and the endogenous phosphorylation sites observed in heart. These data form an interesting starting point to prioritize future targets for drug and biomarker discovery efforts in the cardiac context. Finally, all results are placed into perspective in a concluding summary and outlook.