A NOVEL HIGH RESOLUTION MS APPROACH FOR THE SCREENING OF ADVANCED LIPOXIDATION (ALES) AND ADVANCED GLYCOXIDATION (AGES) END-PRODUCTS INHIBITORS

Several studies indicate that advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs) have a pathogenetic role in the development and progression of different oxidative based diseases including diabetes, chronic renal failure, cardiovascular diseases and neurological disorders. AGEs and ALEs are now considered as promising drug targets and a substantial effort is dedicated to delve the molecular strategies aimed at preventing, reducing or removing these protein oxidation products. A recognized and efficient molecular strategy is based on compounds able to trap the reactive precursors of AGEs and ALEs [1]. Such bioactive compounds can be rationally designed, or fished from libraries or natural matrices, and especially in the latter cases, a suitable analytical method able to evaluate the ability of the tested compounds to inhibit AGEs and ALEs formation is required, and represents the aim of the present study. The method here reported offers the unique advantage of evaluating the efficacy of pure compounds and mixtures as well as raw extracts in inhibiting AGEs and ALEs generated by considering ubiquitin as protein target and a variety of precursors of AGEs and ALEs such as reactive carbonyl species (RCS), including glyoxal, methylglyoxal, 2-hydroxynonenal, acrolein and reducing sugars (glucose and fructose). As a first step, we set-up and implemented a method for the automated injection and quantitative analysis of ubiquitin in native and adducted forms, using a LTQ-Orbitrap XL mass spectrometer coupled to an Ultimate 3000 LC system (Thermo). Ubiquitin AGEs and ALEs adducts were induced by RCSs and sugars incubated at different concentrations and for different time frames. The quantification method automatically measures the areas of the multi-charged peaks of both unmodified and modified ubiquitin. The quantification was linear in the range 0.25-10 \ub5M, with high reproducibility and precision (average CV%=0.12, R2 = 0.99). Ubiquitin AGEs and ALEs were then fully characterized by a bottom-up analysis (nanoLC-MS/MS of digested ubiquitin) and the following covalent modifications were identified: i) Michael adducts on His68, Lys6 and Lys48 upon acrolein and 2-hydroxynonenal incubations; ii) Schiff base adducts on Arg42 and Arg54 upon methylglyoxal incubation. The most reactive RCSs were methylglyoxal and acrolein, while glucose and fructose were the least reactive ones. As a next step, we validated the implemented method by investigating the effect of known inhibitors of AGEs and ALEs formation such as carnosine, hydralazine, aminoguanidine and pyridoxamine. The method was then applied to evaluate the efficacy of natural extracts such as black soybean, rice bran, Panax ginseng and Angelica keiskey. [1] G. Aldini et al. Med Res Rev., 2007, 27(6),817-68