Site-Specific Quantitative Evaluation of the Protein Glycation Product N6-(2,3-Dihydroxy-5,6-dioxohexyl)-l-lysinate by LC−(ESI)MS Peptide Mapping: Evidence for Its Key Role in AGE Formation

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Abstract
Advanced glycation end products (AGEs) contribute to various pathologies associated with the general aging process and long-term complications of diabetes. Involvement of α-dicarbonyl intermediates in the formation of such compounds is firmly established. We now report on the first unequivocal identification of the dideoxyosone N6-(2,3-dihydroxy-5,6-dioxohexyl)-l-lysinate (4) on lysozyme via its quinoxaline derivative N6-(2,3-dihydroxy-4-quinoxalin-2-ylbutyl)-l-lysinate (6), formed by reaction of 4 with o-phenylenediamine (OPD). For accurate quantification of the total content of 6 as well as of glucosepane 5 by LC−(ESI)MS, 13C6-labeled reference compounds were independently synthesized; 5 so far is the only established follow-up product of 4. With an overall lysine derivatization quota of 5%, compound 4 is shown to be a quantitatively important Maillard intermediate of which only about 8‰ are transformed into the cross-link 5. Hence, the major follow-up products of the highly reactive intermediate 4 are yet unknown. The site-specific quantitative evaluation of aminoketose 1 and quinoxaline 6 by LC−(ESI)MS peptide mapping shows that all lysine moieties in lysozyme are in fact modified by these compounds. If an arginine side chain is adjacent to the lysine moiety, transformation of 1 into 4 seems to be favored. The efficient formation and high reactivity of 4 clearly points to its potential as exogenous or endogenous glycotoxin.