NARZĘDZIA CHEMICZNE DO BADANIA ENZYMÓW PROTEOLITYCZNYCH PRZY UŻYCIU CYTOMETRII MASOWEJ

Mass cytometry is one of the newest and most high-throughput technologies that allows for the investigation of complex biological systems at single cell level. It relies on the use of stable metal isotopes as labels of specific cell markers and therefore, allows for simultaneous analysis of more than 40 parameters at single cell level. In order to fully explore the potential of mass cytometry, researchers are trying to develop new experimental setups based on the application of pure metal isotopes in biological studies. The incorporation of antibodies into mass cytometry setups, while extremely selective and well-validated, limits the analysis as it shows the whole protein pool present in the cell. In our group, we developed new technology that allows for the identification of active forms of proteins-the ones that actively participate in cell signaling pathways. Activity-based probes are the most valuable tools for enzyme activity profiling and for years now they have been in the center of the method called Activity-Based Protein Profiling. Classic activity-based probes consist of three parts: a warhead (electrophilic binding group that covalently modifies enzyme active site), linker (specific peptide sequence or non-specific carbon chain) and the fluorescent tag that allows for enzyme detection and localization inside the cell. Spectral properties of commercially available fluorophores allow for the detection of up to dozen different cell parameters, with the use of various techniques such as confocal microscopy or flow cytometry. To increase the number of analyzed parameters, we designed activity-based probes that possess DOTA chelating moiety that is able to trap one metal atom per one probe. The combination of mass cytometry with highly selective activity-based probes allowed for the development of new technology that grants the possibility of multiparametric analysis of complex biological samples such as blood or cancer tissue. The new type of activity-based probes (so-called TOF-probes) incorporate various inhibitor scaffolds designed with HyCoSuL technology (Hybrid Combinatorial Substrate Libraries). These compounds possess a variety of unnatural amino acids in their structures, which significantly increases their selectivity toward proteases of interest.