Quantitative differential proteomics for analysis of posttranslational modifications of proteins

In this work we developed a new and convenient method for high resolution IEF of proteins, which we termed: “daisy chain”. Usually an IEF is accomplished with IPG strips of a desired pH range. For high resolution focusing we are using strips with pH range, which covers only one or two pH units. Thereby the pro-teins, which have isoelectrical point outside of this pH range, are lost. We evalu-ated commercially available IPG strips with consecutive or overlapping pH ranges and connected them serially acidic to basic end, to construct in this way a high resolution IEF-system. For the first time, we showed that a high resolution IEF is possible in such a system and that results were by no means worse than those obtained when the same sample was analyzed on individual single IPGs. The great advantage of our system is that amount of sample used in serial IPG IEF is explicitly lower than when same sample was analyzed on individual single IPGs. This method was subsequently successfully applied to valuable clinical samples from cancer patients and to mitochondrial preparations related to a European project in gerontology. We thus developed a suite of experimental strategies, which adequately address complex biological situations, in particular on the level of protein expression.

Proteomics-driven approach for the detection of breast cancer biomarkers

Breast cancer (BC) is the most often diagnosed cancer entity of women worldwide. No molecular biomarkers are usable in the clinical routine for the early detection of BC. Proteomics is one of the dynamic tools for the successful examination of changes on the protein level. In this thesis different proteomics-based investigations were performed for the detection of protein and autoantibody biomarkers in serum samples of BC and healthy (CTRL) subjects. First, protein levels of candidates from previous profiling studies were investigated via antibody-microarray platform. Three proteins were found in distinct levels in both groups: secretoglobin family 1D member 1, alpha-2 macroglobulin and inter-alpha-trypsin inhibitor heavy chain family member 4. The second part was dedicated to the de novo exploration of potentially immunogenic tumor antigens (TA’s) with immunoprecipitation and Western immunoblotting followed by identification over mass spectrometry. Autoantibody levels were verified in individual serum profiling via the protein microarray platform. Two autoantibody’ cohorts (anti-Histone 2B and anti-Recoverin) were found in different levels in both groups. The findings of this PhD thesis underline deregulated serum protein and autoantibody levels in the presence of BC. Further investigations are needed to confirm the results in an independent study population.