Binder-based technology development
High throughput protein assay systems, based on principles of highly parallel analysis on arrays, are increasingly important for data collection in proteomics. Array technologies to be developed within AFFINOMICS include binder microarrays for establishing protein expression patterns in sera or cell extracts (ULUND, DKFZ), protein arrays for binder characterisation, serum antibody detection and interaction discovery (DKFZ, BBT, KTH), tissue and lysate arrays to analyse cellular protein expression (VTT), and cell arrays using binders to investigate the effects of RNAi (VTT). Novel methods, such as production of protein and binder arrays directly from DNA (BBT, DKFZ), will be expanded to the proteome scale.
A striking molecular analysis technique using binders is proximity ligation (UU), which uses DNA amplification for ultrasensitive and highly specific protein detection and which allows the identification of individual protein molecules and complexes both in solution and in cells in situ. The technology is promising for highly multiplexed analyses, since cross-reactions between pairs of detection reactions can be avoided by appropriate design of the ligation reactions. It is ideally suited to detection, localisation and enumeration of signal transduction and other complexes within cells, and has already demonstrated its potential in measuring protein complexes such as those activated by TGF-β stimulation, and for characterising cell surface receptors and their interactions and phosphorylations. The ability to determine PTMs of the proteins targeted by the binders developed within the project by using modification specific antibodies, e.g. against phosphopeptides, together with proximity ligation assays will enable visualization of functional active sub-compartments of the targeted proteins. Mutations that affect intracellular signalling pathways are likely to result in alterations of protein interactions, and such alterations thus represent promising markers of malignancy. Proximity ligation assays permit such protein interactions to be investigated directly in normal and pathological tissue samples. The ‘snapshot’ data captured by this technique on fixed cells will complement and guide functional experiments on signal transduction pathway analysis, e.g. with intracellularly acting binders in living cells. To date, a major challenge in expanding the utility of methods such as proximity ligation as tools for biomedical research and clinical use has been the availability of specific binders for the target molecules to be detected; the recombinant binders developed within AFFINOMICS will both expand the repertoire of targets and improve the exploitation potential for these technologies. The ability to detect protein interactions and PTMs in clinical specimens by proximity ligation, will introduce completely new functional parameters in tumour biology research and diagnostics. The method will also be invaluable in screening for new compounds interfering with interactions between biomolecules, as the method enables direct measurement of the molecular effect of potential cancer targeting drugs, rather than evaluating indirect measurements of downstream effects such as cell growth or survival.