There is a great need for high-throughput methods to help assign function to the proteins in the cell. In humans, there are ∼30,000 genes that may be expressed in any particular cell type resulting in > 1 million distinct proteins. However, there are few functional proteomic methods to comprehensively study these proteins. One such method to address the cellular function of proteins is CALI (Chromophore-Assisted Laser Inactivation). CALI uses the specificity of antibodies (or other protein binding reagents) and the energy generated from excited dye molecules to specifically damage proteins. FALI (Fluorophore-Assisted Light Inactivation) is a modification of CALI that allows high-throughput protein disruption with a diffuse light source to inactivate samples simultaneously in multiwell plates. FALI is currently being used with large libraries of monoclonal or recombinant antibodies to identify proteins involved in tumor cell invasion, dispersal, and apoptosis. However, new innovations in light-mediated protein inactivation continue to be developed, and provide novel strategies for proteomics. GFP-CALI (Green Fluorescent Protein) and FlAsH-FALI (Fluorescein-Arsenical Helix) are two examples of such innovations. These methods use genetically encoded fusion proteins to direct lightinduced damage to the protein. So far these methods have been used in hypothesis-driven experiments, but they are readily adaptable to high-throughput proteomic screens. In this review, we will describe the current applications of CALI and FALI, and speculate on the future of direct light-induced protein inactivation for functional proteomics.
Keywords: cali, fali, proteomics, high-throughput screens, functional proteomics, cell-based assays, cancer
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