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Deconstruction of activity-dependent covalent modification of heme in human neutrophil myeloperoxidase by multistage mass spectrometry (MS(4)).


Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (1), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by 1 in a labile peptide-heme-peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS(4)) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that 1 was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of 1. The final orientation of 1 perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of 1 by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain.


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  • Authors+Show Affiliations


    Pfizer Worldwide Research, Groton, Connecticut 06340, United States.

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    Biochemistry 51:10 2012 Mar 13 pg 2065-77


    Amino Acid Sequence
    Catalytic Domain
    Chromatography, Liquid
    Crystallography, X-Ray
    Models, Chemical
    Models, Molecular
    Molecular Sequence Data
    Molecular Weight
    Peptide Fragments
    Peptide Mapping
    Tandem Mass Spectrometry

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't
    Research Support, U.S. Gov't, Non-P.H.S.



    PubMed ID