Fluorophore-assisted light inactivation: a high-throughput tool for direct target validation of proteins

Abstract

To exploit advances in proteomics for drug discovery, high-throughout methods for target validation that directly address the cellular roles of proteins are required. To do this, we have characterized fluorophore-assisted light inactivation (FALI) which uses coherent or diffuse light targeted by fluorescein-labeled probes to inactivate specific proteins. We have shown that it is spatially restricted and tested its efficacy in living cells. FALI is efficient using conventional antibodies and single chain variable fragment phage display antibodies (that are compatible with high-throughput applications). We have shown that singlet oxygen is one of the major components required for FALI-mediated damage. The half-maximal radius of damage is approximately 40 A. FALI causes the specific loss of function of beta 1 integrin in HT-1080 fibrosarcoma cells resulting in a reduction in invasiveness. The efficacy of diffuse light sources (such as a desk lamp) with FALI to inactivate many samples in parallel provides an inexpensive, high-throughput method of wide general applicability for functional proteomics.