Top-down and bottom-up mass spectrometric characterization of human myoglobin-centered free radicals induced by oxidative damage.
In an effort to determine the utility of top-down mass spectrometric methodologies for the characterization of protein radical adducts, top-down approaches were investigated and compared to the traditional bottom-up approaches. Specifically, the nature of the radicals on human myoglobin induced by the addition of hydrogen peroxide and captured by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was investigated. The most abundant ion observed in the electrospray mass spectrum of this reaction mixture corresponds in mass to the human myoglobin plus one DMPO molecule. In addition, a second ion of lower abundance is observed, which corresponds to a second DMPO molecule being trapped on myoglobin. Top-down analyses using Fourier transform ion cyclotron resonance mass spectrometry can be used to characterize proteins and, thus, were performed on several different charge-state ions of both the native and the mono-DMPO nitrone adduct of human myoglobin. Data produced from the top-down analyses are very complex yet information rich. In the case of DMPO-modified human myoglobin, the top-down data localized the DMPO spin trap to residues 97-110 of the myoglobin. The observation of the y43+5 fragment ion arising from C-terminal cleavage to the cysteine-110 residue in the MS/MS spectrum of DMPO-modified myoglobin and not in the unmodified myoglobin implicates a change to this residue, specifically, DMPO adduction. On the other hand, using the traditional bottom-up approach of peptide mapping and MS sequencing methodologies, two DMPO radical adducts on human myoglobin were identified, Cys-110 and Tyr-103. The bottom-up approach is more proven and robust than the top-down methodologies. Nonetheless, the bottom-up and top-down approaches to protein characterization are complementary rather than competitive approaches with each having its own utility.
Deterding
Leesa J
LJ
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, MD F0-03, Research Triangle Park, North Carolina 27709, USA. deterdi2@niehs.nih.gov
Bhattacharjee
Suchandra
S
Ramirez
Dario C
DC
Mason
Ronald P
RP
Tomer
Kenneth B
KB
D003497:Cyclic N-Oxides; D005609:Free Radicals / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide; D013058:Mass Spectrometry / Q000379:methods*; D009211:Myoglobin / Q000737:chemistry*; D018384:Oxidative Stress
D003497:Cyclic N-Oxides; D005609:Free Radicals; D009211:Myoglobin; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D006861:Hydrogen Peroxide