Using tandem mass spectrometry to quantify site-specific chlorination and nitration of proteins: model system studies with high-density lipoprotein oxidized by myeloperoxidase.
Protein oxidation is implicated in atherogenesis and other inflammatory conditions. Measuring levels of chlorinated and nitrated proteins in biological matrices serves as a quantitative index of oxidative stress in vivo. One potential mechanism for oxidative stress involves myeloperoxidase, a heme protein expressed by neutrophils, monocytes, and some populations of macrophages. The enzyme uses hydrogen peroxide to generate an array of cytotoxic oxidants, including hypochlorous acid (HOCl), a potent chlorinating intermediate, and nitrogen dioxide radical, a reactive nitrogen species (RNS). One important target may be high-density lipoprotein (HDL), which is implicated in atherogenesis. This chapter describes liquid chromatography-tandem mass spectrometric methods for quantifying site-specific modifications of proteins that have been oxidized by HOCl or RNS. Our studies center on apolipoprotein A-I, the major HDL protein, which provides an excellent model system for investigating factors that target specific residues for oxidative damage. Our approach is sensitive and rapid, applicable to a wide array of posttranslational modifications, and does not require peptides to be derivatized or labeled with an isotope.
Shao
Baohai
B
Department of Medicine, University of Washington, Seattle, Washington, USA.
Heinecke
Jay W
JW
D000595:Amino Acid Sequence; D000818:Animals; D002713:Chlorine / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D054879:Halogenation; D006801:Humans; D008075:Lipoproteins, HDL / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009574:Nitro Compounds / Q000032:analysis* / Q000737:chemistry; D009195:Peroxidase / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D008075:Lipoproteins, HDL; D009574:Nitro Compounds; D002713:Chlorine; D009195:Peroxidase