Analytical strategies for the identification and characterization of protein adducts with HNE and related compounds

Advanced Lipoxidation Endproduct (ALEs) are modified proteins that can act as pathogenic factors in several chronic diseases, like diabetes and cardiovascular diseases [1]. These covalent adducts belong to a heterogeneous class of compounds derived from the protein adduction by reactive carbonyl species (RCS), which are generated upon lipid peroxidation. A similar class of compounds, Advanced Glycation Endproducts (AGEs), exhibit the same damaging effects as ALEs, partly due to binding to the Receptor for Advanced Glycation End products (RAGE). Using this receptor as a stationary phase for affinity chromatography, modified proteins could be potentially entrapped and enriched from any sample, to identify and characterize the origin of the modification. Applying this innovative approach, it has been shown already that AGEs can be captured, enabling the full characterization of the adducted moieties and site of modification using a bottom-up approach [2]. In order to validate this strategy for ALEs, and with the aim to understand whether ALEs are also binder of RAGE, fully characterized ALEs were produced in-vitro, by incubating human serum albumin (HSA) with glyoxal (GO), methylglyoxal (MG), 4-hydroxynonenal (HNE), acrolein (ACR) and malondialdehyde (MDA). The formation of ALEs was confirmed using a top-down MS approach by direct infusion on a triple-quadrupole mass spectrometer and the modifications and sites of adduction fully characterized by a bottom-up approach. The in-vitro produced ALEs were then subjected to VC1 Pull-Down relying on magnetic beads bound to VC1, the domain of RAGE necessary for binding, which can easily be separated. ALEs will be retained by VC1 and unbound protein can be easily removed, enriching ALEs in the sample. After binding, ALEs are eluted from the magnetic beads and subjected to GeLC-MS/MS to identify and localize the modification. Data obtained using this method were analysed using a targeted approach based on setting known modifications. Results obtained using VC1 Pull-Down were compared to the results of the in-solution digested ALEs and showed that ALEs containing a cyclic moiety induced by the modifications, are better retained by VC1, including pyrimidine, pyridine, pyraline and imidazolone adducts. Another observation is the binding of ALEs containing a carboxy-derivative, since these adducts exhibit a negative charge and increases the binding specificity to VC1, which has a positive charge. Semi-quantitative analysis also showed an enrichment of ALEs from VC1 Pull-Down, compared to unenriched sample. Results showed that VC1 can be used as a stationary phase to selectively enrich ALEs, depending on the structure and nature of the modification. Different applications of this technique are underway to identify and characterize ALEs and AGEs from samples of patients affected by diseases involving oxidative stress. In conclusion, we have found that besides AGEs, also ALEs are RAGE binders. ALEs involvement in the RAGE dependent proinflammatory cascade is currently under investigation.