title: 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 modification in different age-related pathological processes. To facilitate the detection of these biomolecules, we developed a strategy in which the purified enzyme (or protein) of interest is first treated with authentic isoLG in vitro to evaluate whether it contains reactive lysine residues prone to modification 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 modification in a biological sample. In this chapter, we describe the conditions for the in vitro isoLG modification assay and how to use mass spectrometry to identify the isoLG-modified peptides and amino acid residues. Our studies were carried out on cytochrome P450 27A1, an important metabolic enzyme, and utilized iso[4]levuglandin E2 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 modified peptides. The developed protocol could be applied to characterization of other enzymes/proteins and other types of unconventional posttranslational protein modification.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH, 44106, USA.
authors.forename: Casey D
authors.initials: CD
authors.lastname: Charvet
authors.forename: Irina A
authors.initials: IA
authors.lastname: Pikuleva
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D053493:Cholestanetriol 26-Monooxygenase / Q000378:metabolism*; D002853:Chromatography, Liquid; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D059748:Proteolysis; D012984:Software
chemical_list: D000596:Amino Acids; D005231:Fatty Acids, Unsaturated; C526910:iso(4)levuglandin E2; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase