Using tandem mass spectrometry to quantify site-specific chlorination and nitration of proteins: model system studies with high-density lipoprotein oxidized by myeloperoxidase

Methods Enzymol. 2008:440:33-63. doi: 10.1016/S0076-6879(07)00803-8.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Chlorine / analysis
  • Chlorine / chemistry
  • Chlorine / metabolism*
  • Chromatography, Liquid
  • Halogenation*
  • Humans
  • Lipoproteins, HDL / chemistry*
  • Lipoproteins, HDL / genetics
  • Lipoproteins, HDL / isolation & purification*
  • Lipoproteins, HDL / metabolism
  • Models, Chemical*
  • Molecular Sequence Data
  • Nitro Compounds / analysis*
  • Nitro Compounds / chemistry
  • Peroxidase / chemistry*
  • Peroxidase / metabolism
  • Spectrometry, Mass, Electrospray Ionization
  • Tandem Mass Spectrometry

Substances

  • Lipoproteins, HDL
  • Nitro Compounds
  • Chlorine
  • Peroxidase