Development of Fast Photochemical Oxidation of Proteins for In Vivo Modification in Caenorhabditis elegans

Abstract

Caenorhabditis elegans, part of the nematode family, are used as model systems for many human diseases including those involving protein misfolding such as Parkinson's disease. Structural studies on C. elegans strains expressing the aggregate forms of the proteins involved in these diseases would shed light on how these aggregate structures lead to disease. The protein footprinting method fast photochemical oxidation of proteins (FPOP) is especially suited for these in vivo studies because of the irreversible nature of the modification. FPOP utilizes hydroxyl radicals to oxidatively modify solvent accessible sites in proteins. Radicals are generated via hydrogen peroxide photolysis by a 248 nm excimer laser. Our lab has performed preliminary studies that indicate that FPOP can be used to study protein structure directly in C. elegans. We have designated this new iteration of the method in vivo FPOP (IV-FPOP). Here, we describe the use of penetration enhancers in order to increase the number of oxidatively modified proteins. C. elegans can uptake hydrogen peroxide directly through their skin and the addition of penetration enhancers (i.e. DMSO) will aid with hydrogen peroxide uptake. The viability of the worms in the presence of hydrogen peroxide and these enhancers were also tested. LC-MS/MS analysis revealed and increase in oxidatively modified proteins by IV-FPOP when worms were treated with penetration enhancers. This work further demonstrates the efficacy of IV-FPOP for studying proteins directly in an animal model.