Structural analysis of naphthoquinone protein adducts with liquid chromatography/tandem mass spectrometry and the...

The relative reactivities of various naphthoquinone isomers (1,4-, 1,2- and 2-methyl-1,4-naphthoquinone) to two test proteins, apomyoglobin and human hemoglobin, were evaluated via liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The structural characterization of the resulting adducts was also obtained by LC/ESI-MS analysis of the intact proteins. The reactive sites of apomyoglobin and human hemoglobin with 1,4-naphthoquinone and 1,2-naphthoquinone were also identified through characterization of adducted tryptic peptides by use of high-pressure liquid chromatography/electrospray ionization with tandem mass spectrometry (HPLC/ESI-MS/MS), TurboSEQUEST, and the scoring algorithm for spectral analysis (SALSA). Four adducted peptides, which were formed by nucleophilic addition of a lysine amino acid residue to 1,4-naphthoquinone, were also identified, as was an adducted peptide from incubation of 1,2-naphthoquinone with apomyoglobin. In the case of incubation of human hemoglobin with the two naphthoquinones, two adducted peptides were identified from the N-terminal valine modification of the alpha and beta chains of human hemoglobin. The adducted protein formation may imply that naphthalene produces its in vivo toxicity through 1,2- and 1,4-naphthoquinone metabolites reacting with biomolecular proteins.