Top-down and bottom-up mass spectrometric characterization of human myoglobin-centered free radicals induced by oxidative damage

Abstract

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.

Figure 1

FTICR MS data obtained from the reaction of human myoglobin with hydrogen peroxide in the presence of DMPO. A) Human myoglobin and B) Human myoglobin plus DMPO and H₂O₂. An expanded view of the +15 charge state ion is shown in the insets.

Figure 2

Q-Tof/CAD MS/MS spectra of the (M+18H)¹⁸⁺ ion of A) m/z 948.4 of human myoglobin and B) m/z 954.7 of human myoglobin-DMPO nitrone adduct.

Figure 3

Expanded mass range from m/z 985–1005 from the Q-Tof/CAD MS/MS spectra of the (M+18H)¹⁸⁺ ion of A) m/z 948.4 of human myoglobin and B) m/z 954.7 of human myoglobin-DMPO nitrone adduct.

Figure 4

Top down map of the cleavages observed in the tandem mass spectra of human myoglobin. Fragment ions in red were observed in the Q-Tof CAD MS/MS data. Fragment ions in black were observed in the FTICR IRMPD and/or IRMPD/ECD data. Underlined residues are observed as internal fragmentations in the IRMPD spectra.

Figure 5

FTICR IRMPD MS/MS spectra of the (M+15H)¹⁵⁺ ions of A) human myoglobin and B) human myoglobin-DMPO nitrone adduct. Ions labeled as “*I” are internal fragment ions and ions labeled as “B” are background ions.

Figure 6

Overlay of FTICR IRMPD MS/MS spectra of the (M+15H)¹⁵⁺ ions of human myoglobin (black line) and human myoglobin-DMPO nitrone adduct (red line). Expanded mass range of A) m/z 1282.5 to 1285.5 and B) m/z 945.0 to 957.5.

Figure 7

FTICR IRMPD/ECD MS/MS spectrum of the (M+15H)¹⁵⁺ ion of human myoglobin.

Figure 8

A) Deconvoluted ESI/MS/MS spectrum of the (M+3H)³⁺ ion of m/z 675.35 which corresponds in mass to tryptic peptide T17 plus one DMPO adduct of human myoglobin. B) Deconvoluted ESI/MS/MS spectrum of the (M+3H)³⁺ ion of m/z 712.36 which corresponds in mass to tryptic peptide T17 plus two DMPO adducts of human myoglobin.

Figure 9

Positions of the amino acid residues in human myoglobin treated with hydrogen peroxide and trapped by DMPO as determined by mass spectrometry. Figure was generated from the x-ray crystal structure of the human myoglobin double mutant, K45R and C110A (46). The backbone of myoglobin is shown in cyan. The heme is shown in red, Ala-110 (corresponding to Cys-110) is shown in yellow and Tyr-103 is shown in purple.