Analysis of non-protein amino acids as specific markers of protein oxidation: the use of N(O,S)-ethoxycarbonyl ethyl ester and N(O,S)-ethoxycarbonyl trifluoroethyl ester derivatives and GC-MS

Oxidative modification of proteins is widely regarded as a crucial event in the pathogenesis of various inflammatory and metabolic diseases. In this line, a sensitive and specific GC-MS methodology using either N(O,S)-ethoxycarbonyl ethyl amino acid esters (ECEE) or N(O,S)-ethoxycarbonyl trifluoroethyl amino acid esters (ECEE-F3) for rapid and sensitive determination of modified amino acid side chain residues as specific oxidation markers in proteins has been developed.
Both ECEE and ECEE-F3 derivatives are formed by the unlabored reaction of amino acids with ethyl chloroformate plus ethanol or trifluoroethanol plus pyridine. The key steps of the methodology involve enzymatic hydrolysis of target proteins to prevent decomposition of oxidation products during hydrolysis and uniquely rapid derivatization of modified amino acids completing sample preparation for GC within a few minutes in aqueous solution at room temperature. The use of this methodology for assessing (glyc)oxidative damage in low density lipoprotein apolipoprotein B-100 (apoB-100) recovered from human plasma and various inflammatory compartments has been demonstrated. The observations provided quantitative chemical evidence for (glyc)oxidative processes in several inflammatory and metabolic diseases.