Design of New Probes for Oxidized Amino Acids Localization

Abstract : Protein carbonyls (PC) are oxidative damage observed in many diseases. Proteins are possibly the most immediate vehicle for inflicting oxidative damage on cells because they are often catalysts [1]. A fluorometric and UV-absorption method have been developed to quantify PC in blood and tissues samples by labeling with two hydrazines: 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) and dinitrophenylhydrazine (DNPH), respectively [2,3]. These methods are based on the selective hydrazone formation between the carbonyls group of oxidized protein to yield a strong fluorescent/UV-absorption adduct that is then quantified. Here, we will describe our study on NBDH's and DNPH's derivatives to generate new PC-probes bearing an alkyne moiety. In a first step, the hydrazine moiety reacted specifically with protein carbonyls and in a second step, a click reaction was performed between the alkyne moiety and a cleavable resin [4] to yield a PC's enrichment. Theses probes have been explored on oxidized bovine serum albumin (OxBSA) for PC-labeling, and the possible sites of oxidation of isolated labelled PC will be studied by LC-MS and proteomics experiments. References 1. Dalle-Donne, I.; Rossi, R.; Giustarini, D.; Milzani, A.; Colombo, R. Protein carbonyl groups as biomarkers of oxidative stress. Clin. Chim. Acta 2003, 329, 23-38. 2. Stocker, P.; Ricquebourg, E.; Vidal, N.; Villard, C.; Lafitte, D.; Sellami, L.; Pietri, S. Fluorimetric screening assay for protein carbonyl evaluation in biological samples. Anal. Biochem. 2015, 482, 55-61. 3. Vidal, N.; Cavaille, J.P.; Graziani, F.; Robin, M.; Ouari, O.; Stocker, P. High throughput assay for evaluation of reactive carbonyl scavenging capacity. Redox Biology 2014, 2, 590-598. 4. Sibbersen, C.; Lykke, L.; Gregersen, N.; Jørgensen, K.A.; Johannsen, M. A cleavable azide resin for direct click chemistry mediated enrichment of alkyne-labeled proteins.