Advanced methodology combining UPLC-MS, isotopic labelling and H/D exchanges reveals three tyrosine-tyrosine cross-links induced by oxidative radicals evolving to at least four dimeric structures.

Di-tyrosine is one of the major protein cross-links involved in a large number of neurodegenerative or ageing-related diseases. Recently, no less than four different di-tyrosine bridge isomers have been highlighted while only two structures are characterized at the moment in the literature. In this study, the four dimers were produced by radiolytical-induced oxidation. Although the abundance of these additional dimers precluded the use of NMR or other structural characterization methods, we propose a new methodology combining UPLC-MS analysis, specific deuterium labelling and isotopic (H/D) exchanges with the solvent. Thus, we were able to identify three different covalent cross-links and propose different new original di-tyrosine structures based on double Michael additions, leading to tetracyclic products. Absorption and fluorescence characterizations of the four species were performed and consolidate our proposal.