Detecting oxidative modification of biomolecules with isotope dilution mass spectrometry: Sensitive and quantitative assays for oxidized amino acids in proteins and tissues

Abstract

This chapter describes sensitive and quantitative assays for measuring o-tyrosine, m-tyrosine, o,o'-dityrosine, 3-nitrotyrosine, and 3-chloro-tyrosine in proteins and tissues. The methods combine gas chromatography (GC) with isotope dilution negative-ion electron capture mass spectrometry (MS). GC/MS has enabled to determine the relative amounts of oxidized amino acids in proteins and lipoproteins oxidized in vitro. This approach also quantifies levels of modified amino acids in atherosclerotic lesions, inflammatory tissues, and tissue proteins of aging animals. The observations indicate that the quantitative evaluation of protein oxidation products by GC/MS is a powerful tool for identifying pathways that promote oxidative protein modification in vivo. The specificity, sensitivity, and precision of isotope dilution GC/MS to identify products of specific oxidation pathways makes this approach a remarkably powerful technique for this purpose. Development of further mass spectrometric methods for detecting specific molecules in vivo should provide important insights into the involvement of particular oxidative pathways in the onset and progression of a variety of human diseases.