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

Oxidative modification of proteins can interfere with critical cellular functions, and is widely regarded as a crucial event in the pathogenesis of various diseases ranging from rheumatoid arthritis to atherosclerosis and cancer. The mechanisms of formation and the nature of the altered amino acid side chain residues formed on proteins subjected to oxidant attack are reviewed. In this line, a new GC-MS method using N(O,S)-ethoxycarbonyltrifluoroethyl amino acid esters (ECEE-F₃) for rapid and sensitive determination of 3-chlorotyrosine, 5-hydroxy-2-aminovaleric acid (HAVA), and 6-hydroxy-2-aminocaproic acid (HACA) in proteins is discussed. 3-Chlorotyrosine is a highly specific marker of myeloperoxidase-catalyzed protein oxidation, whereas HAVA and HACA are specifically formed by metal-catalyzed oxidation processes. ECEE-F₃ derivatives are formed by the unlabored reaction of amino acids with ethylchloroformate plus trifluoroethanol plus pyridine. The key steps of the methodology employed are i) enzymatic hydrolysis of target proteins to prevent decomposition of oxidation products during hydrolysis and ii) an uniquely rapid derivatization of amino acids completing sample preparation for GC within a few minutes in aqueous solution at room temperature. Femtomole levels of 3-chlorotyrosine, HAVA, and HACA can be reproducible measured in different protein preparations subjected to oxidative damage in vitro. Furthermore, the use of these stable products of protein amino acid side chain oxidation as potential markers for assessing oxidative damage in patients with inflammatory and metabolic disorders ex vivo and in vivo is demonstrated. Evidence is presented to show that the pattern of products detected may yield information as to the nature of the original oxidative insult.