Mass spectrometry detection of isolevuglandin adduction to specific protein residues

Abstract

The aging process seems to be associated with oxidative stress and hence increased production of lipid peroxidation products, including isolevuglandins (isoLGs). The latter are highly reactive γ-ketoaldehydes which can form covalent adducts with primary amino groups of enzymes and proteins and alter the properties of these biomolecules. Yet little is currently known about amino acid-containing compounds affected by isoLG modifi cation in different age-related pathological processes. To facilitate the detection of these biomolecules, we developed a strategy in which the purifi ed enzyme (or protein) of interest is fi rst treated with authentic isoLG in vitro to evaluate whether it contains reactive lysine residues prone to modifi cation with isoLGs. The data obtained serve as a basis for making the “GO/NO GO” decision as to whether to pursue a further search of this isoLG modifi cation in a biological sample. In this chapter, we describe the conditions for the in vitro isoLG modifi cation assay and how to use mass spectrometry to identify the isoLG-modifi ed peptides and amino acid residues. Our studies were carried out on cytochrome P450 27A1, an important metabolic enzyme, and utilized iso[4]levuglandin E 2 as a prototypical isoLG. The isoLG-treated cytochrome P450 was subjected to proteolysis followed by liquid chromatography-tandem mass spectrometry for peptide separation and analysis by Mascot, a proteomics search engine, for the presence of modifi ed peptides. The developed protocol could be applied to characterization of other enzymes/proteins and other types of unconventional posttranslational protein modifi cation.