Identification and characterization of pro-inflammatory AGEs and ALEs by VC1-affinity chromatography and mass spectrometry

The Receptor for AGEs (RAGE), is a membrane receptor binding different ligands sharing a common pattern, including Advanced Glycation/Lipoxidation Endproducts (AGEs/ALEs). The precise mechanism and structural requirements by which AGEs/ALEs interact with RAGE still remain to be elucidated. We have set-up a selective enrichment procedure for AGEs/ALEs based on RAGE ectodomain (VC1) as a stationary phase. The technique was validated using in-vitro produced AGEs/ALEs: HSA as a model protein and reactive carbonyl species and reducing sugars as modifying agents. Generated AGEs/ALEs were successfully entrapped and then fully characterized by Mass Spectrometry (MS). A cyclic moiety was identified as a common structure of the retained AGEs and ALEs, and its involvement in RAGE binding was clarified by molecular modelling studies. Currently, the assay is used to enrich AGEs/ALEs present in different biological matrices, such as oxidized human plasma and clinical samples, for their characterization by MS. The advantage of the herein described technique is to entrap RAGE ligands, independently of their structures, and without the use of any covalent derivatization process. The pro-inflammatory activity of characterized AGEs/ALEs was then tested in a cellular model overexpressing RAGE.