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LC/MSMS STUDY OF BENZO[A]PYRENE-7,8-QUINONE ADDUCTION TO GLOBIN TRYPTIC PEPTIDES AND N-ACETYLAMINO ACIDS
Citation:
WINNIK, W. M., N. BALU, W. PADGETT, AND S. NESNOW. LC/MSMS STUDY OF BENZO[A]PYRENE-7,8-QUINONE ADDUCTION TO GLOBIN TRYPTIC PEPTIDES AND N-ACETYLAMINO ACIDS. Presented at American Society for Mass Spectrometry, Seattle, WA, May 28 - June 01, 2006.
Description:
Benzo[a]pyrene-7,8-quinone (BPQ) is regarded as a reactive genotoxic compound enzymatically formed from a xenobiotic precursor benzo[a]pyrene-7,8-diol by aldo-keto-reductase family of enzymes. Because BPQ, a Michael electrophile, was previously shown to react with oligonucleotides and glutathione, BPQ might also attack other nucleophilic cell components such as regulatory proteins. Quinone enzymes participate in redox cycling reactions in mitochondria, hence BPQ might interfere with oxidative phosphorylation processes and produce toxic effects by the mechanisms of oxidative stress. In this work, products of the BPQ reaction with globin peptides are studied mainly by LC/+/nanospray/MSMS and also by NMR. The MSMS fragmentation pattern of the adducts is a base for future analytical studies in vivo.
BPQ was prepared using a method described by Harvey and Fu (1978, In Polycyclic Hydrocarbons and Cancer, Gelboin, H, and Tso, P, Eds, pp 133-165) from trans-7,8-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Molar excess of BPQ was dissolved in DMSO or DMF in phosphate buffer and reacted at pH 7.2 with small biologically-relevant nucleophiles: glutathione, N-acetylcysteine, N-acetyllysine, and also with relatively larger target protein and the tryptic peptides of globin: GTFATLSELHCDK, VHLTPEEK, and VLSPADK. LC/+/nanospray/MSMS experiments were performed using an ion-trap mass spectrometer and a nanospray column operating at a flowrate of 250-300 nl/min. P-MOD shareware software (graciously provided by Dr. Liebler) aided in the interpretation of the MSMS spectra of adducted peptides.
A LC/MS chromatogram and Zoom-ScanTM spectra of the room-temperature reaction synthesis products of BPQ with a tryptic globin peptide GTFATLSELHCDK shows a spectrum of a major adduct characterized by the mass increase of 282 Da, which is in agreement with the addition of C20H10O2 from BPQ. A MSMS spectrum indicates the presence of a BPQ Michael o-dihydroquinone adduct situated on the sulphydryl group of cysteine. This spectrum displays besides the correct series of the "b" and the "y" ions, prominent peaks of singly and doubly-charged ions corresponding to the loss of the mass of 282 Da from the precursor ion. A LC/MSMS chromatogram of an analogous reaction carried out at 37C shows two additional major LC peaks, which were identified based on their MSMS spectra as corresponding to: (1) GTFATLSELHC[+48 Da]DK possessing an oxidized cysteine, and an adduct of 276 Da located on the N-terminus G[+276 Da]TFATLSELHCDK, suggesting the presence of an oxidized form of a BPQ adduct. Various BPQ adducts were observed when the VHLTPEEK and VLSPADK globin peptides were reacted with BPQ at 37C and 55C. The VH[+282 Da]LTPEEK adduct was the major adduction product. This adduct was proposed based on the strong agreement between the MSMS fragmentation pattern of the singly and doubly charged ions, a Zoom-ScanTM spectrum and an expected "b" and the "y" ion series. A valine adduct of BPQ and a molecule of water (282 Da plus 18 Da), V[+300]HLTPEEK, and a minor BPQ adduct in a quinone form, V[+280 Da]HLTPEEK, were also postulated based on their MSMS fragmentation patterns. C-terminus lysine-adducts VLSPADK[+300 Da] and VLSPADK[+280 Da] were proposed for the reaction of BPQ with VLSPADK.