Advancement of an antibody microarray platform for the simultaneous profiling of protein expression and multiple post-translational modifications

The list of functions that post-translational modifications (PTMs) have in the human organism and for cellular processes, highlights their biological relevance. Consequently, the development of assays for the analysis of these modifications, are of high interest in order to be able to investigate different diseases. Antibody microarrays offer a promising approach for investigating PTMs across a wide range of target proteins and sample types using small sample quantities. They enable a sensitive detection of differences in protein abundance and PTMs in a parallel and high-throughput analysis. Currently, available tools for PTM analysis on antibody microarrays use only a parallel detection of one specific PTM in combination with the analysis of the protein abundance. The central objective of this thesis was the development of a single assay capable of analysing two specific PTMs in combination with the respective protein abundance. Two antibody microarray types were designed and produced to establish an appropriate platform for the development of a dual-PTM assay. In this thesis, acetylation and phosphorylation were selected as the PTMs for the assay development. After conducting multiple feasibility studies and adapting different incubation parameters, the developed assay was used to perform a proof-of-concept study. It focused on the comparison of the newly developed dual-PTM assay with standard single-PTM assays. Furthermore, the identification of biomarkers to distinguish biological differences in patient derived samples was the second aim of the concept study. To this end ovarian cancer ascites samples from 26 different patients were selected. These patients were previously classified into responder and non-responder to two different treatments. The first treatment was with a superantigen, produced by Staphylococcus aureus (SEA). The second treatment was with Ipilimumab, an immune checkpoint Inhibitor, blocking the cytotoxic T-lymphocyte protein 4 (CTLA-4) receptor, which is expressed on T lymphocytes, regulating activation in an early stage of immunoreaction. The aim was to validate the newly developed assay and at the same time identify biomarkers or combinations of biomarkers that were able to predict a response to the two different treatments. Comparing the performance of single-PTM and dual-PTM assay, no major differences between the two assays were observed. Differential abundant proteins as well as phosphorylation-dependent differences were identified between responder as well as nonresponder. No differential acetylation was detected, neither with the single-PTM assay nor with the dual-PTM assay. To integrate the data from this proof-of-concept study into a broader biological context, data of the dual-PTM assay were used for the identification of biomarker combinations for both treatments. Linear support vector machines were used to select combinations for each treatment, two biomarker panels were selected, incorporating both protein abundance and phosphorylation status. These panels are examined in their biological context. In future the adaption of different PTM combinations in one assay would be an important step in the field of biomarker development which would be of meaningful interest for science. This emphasizes the biological significance of the development of the dual-PTM assay.