High resolution mass spectrometric strategies for studying AGEs inhibitors and RAGE antagonists

AGEs are involved in the onset and progression of different oxidative based diseases and their inhibition, together with the blockade of the AGEs-RAGE interaction, represents a promising drug target. In order to exploit AGEs and RAGE as drug targets, validated analytical methods able to screen compounds acting as AGEs inhibitors or as RAGE antagonists are required. An analytical platform based on high-resolution mass spectrometry (MS) which permits the evaluation of the ability of the tested compounds to inhibit AGEs-ALEs formation and their interaction with RAGE is here reported. Testing AGEs/ALEs inhibitors: the method we set-up offers the unique advantage of evaluating the efficacy of pure compound, mixture and raw extract on inhibiting AGEs and ALEs generated by incubating ubiquitin as protein target with reactive carbonyl species (RCS) such as glyoxal, methylglyoxal, 2-hydroxynonenal, acrolein, or reducing sugars (glucose and fructose). The method is based on automated injection and quantitative analysis of ubiquitin in native and adducted forms, using an Orbitrap mass spectrometer. The method was validated by investigating the effect of known inhibitors of AGEs and ALEs formation such as carnosine, hydralazine, aminoguanidine and pyridoxamine as well as of some natural extracts. Testing RAGE antagonists: a MS method was firstly set up to study the non-covalent interactions between ligands and recombinant sRAGEs (V1-C1), representing the ligand binding domain of RAGE. The V1-C1 protein target was expressed in E. coli and the ligand-protein binding properties (stoichiometry and Kd values) were determined by a high-resolution mass spectrometric (orbitrap) approach carried out in not-denaturating conditions and using a static nano-ESI source. The method was then validated by using well known low molecular weight sRAGE ligands and the Kd values were in line with those previously reported by fluorescence titration experiments.