Objective

The analysis of cellular signalling cascades has proven to be a valuable approach for understanding the processes that underlay basic cellular functions and allow the detection of changes that are important during disease. The profiling of central signalling pathways requires the detection of changes in protein expression and differences in protein modification or activation. Our novel DigiWest technology combines the principles of Western blotting with a multiplexed Luminex bead array as a readout system. Thereby, the system allows the generation of information on the expression and modification of hundreds of proteins.

Using this approach, we analysed 24 fresh frozen tumour specimens from relapsed vs cured ovarian cancer patients directly on the protein level. By employing nearly 500 antibodies a comprehensive characterisation of activation states of signalling cascades was possible and expression levels of tumor and metastasis marker proteins were determined. Data analysis revealed a cluster of proteins that discriminates relapsed from cured patients via differential activation states of central signal transduction pathways, such as MAPK and NFkB signaling. Based on this, we extracted a signature of eight proteins and protein modifications whose differential expression was sufficient to clearly distinguish relapsed from cured patients, and which therefore represent promising biomarker candidates.