Effects of Fe(II)/H2O2 oxidation on ubiquitin conformers measured by ion mobility-mass spectrometry

Abstract

Oxidative modifications can have significant effects on protein structure in solution. Here, the structures and stabilities of oxidized ubiquitin ions electrosprayed from an aqueous solution (pH 2) are studied by ion mobility spectrometry-mass spectrometry (IMS-MS). IMS-MS has proven to be a valuable technique to assess gas phase and in many cases, solution structures. Herein, in vitro oxidation is performed by Fenton chemistry with Fe(II)/hydrogen peroxide. Most molecules in solution remain unmodified, whereas ∼20% of the population belongs to an M+16 Da oxidized species. Ions of low charge states (+7 and +8) show substantial variance in collision cross section distributions between unmodified and oxidized species. Novel and previously reported gaussian conformers are used to model cross section distributions for +7 and +8 oxidized ubiquitin ions, respectively, in order to correlate variances in observed gas-phase distributions to changes in populations of solution states. Based on gaussian modeling, oxidized ions of charge state +7 have an A-state conformation which is more populated for oxidized relative to unmodified ions. Oxidized ubiquitin ions of charge state +8 have a distribution of conformers arising from native-state ubiquitin and higher intensities of A- and U-state conformers relative to unmodified ions. This work provides evidence that incorporation of a single oxygen atom to ubiquitin leads to destabilization of the native state in an acidic solution (pH ∼2) and to unfolding of gas-phase compact structures.

Figure 1

High resolution mass spectra of (a) unmodified and (b) oxidized ubiquitin obtained upon ESI-LTQ-Orbitrap MS analysis. The insets show zoomed-in regions of the +10 charge state ions.

Figure 2

CID MS/MS spectra obtained upon isolation (±1 m/z) of the +10 charge state ions of (a) unmodified and (b) oxidized ubiquitin species. The assigned b- and y-type fragment ions are listed in the figure. The insets show the noted magnification of the m/z range 240–400 which highlight the b₂ and b₃ ions.

Figure 3

(a) Two-dimensional drift time (m/z) contour plot for the Fe(II)/H₂O₂-induced oxidized ubiquitin electrosprayed from a solution of water and formic acid (pH 2; see Experimental). Ions of charge states from +7 to +13 are observed and each charge state has been provided as labels. To observe the features of charge states +7 and +8 more clearly, corresponding regions (marked by white boxes) have been zoomed in and displayed in (b) and (c), respectively. The unmodified ions are labeled as [M+7H]⁷⁺ (b) and [M+8H]⁸⁺ (c); the oxidized ions are labeled as [M+O+7H]⁷⁺ (b) and [M+O+8H]⁸⁺ (c). The insets in (b) and (c) show the drift time distributions for the corresponding ubiquitin species as labeled, which are normalized by the integrated peak intensity.

Figure 4

Collision cross section (ccs) distributions for Fe(II)/H₂O₂-treated ubiquitin of charge states +7 (a) and +8 (b) electrosprayed from different water:formic acid solutions. The solution pH is labeled for each of the distributions. Distributions for unmodified and oxidized ubiquitin are plotted as black dashed lines and red solid lines, respectively. The distributions are normalized by the integrated peak intensity.

Figure 5

Gaussian models of collision cross section (ccs) distributions for unprocessed ubiquitin of charge state +7 electrosprayed from two solution conditions (100:0 and 50:50 water:methanol, pH ~2 adjusted by formic acid). The solution conditions have been labeled. The experimental data (normalized) are drawn as black circles, the Gaussian distributions employed in the modeling are depicted as blue, green and pink solid lines, representing the N, A, and U state, respectively, and the sums of the Gaussian functions are shown as red dashed lines.

Figure 6

Gaussian models of collision cross section (ccs) distributions for Fe(II)/H₂O₂-treated ubiquitin of charge states +7 (a) and +8 (b) electrosprayed from a solution of water and formic acid (pH 2; see Experimental). The ccs distributions of unmodified ubiquitin are labeled as [M+7H]⁷⁺ and [M+8H]⁸⁺ for charge states of +7 and +8, respectively. The ccs distributions of oxidized ubiquitin are labeled as [M+O+7H]⁷⁺ and [M+O+8H]⁸⁺ for charge states of +7 and +8, respectively. The solution conditions (water:methanol 100:0, pH ~2 adjusted by formic acid) have been labeled. The experimental data (normalized) are shown as black circles, the Gaussian distributions employed in the modeling are drawn as blue, green and pink solid lines, representing the N, A, and U state, respectively, and the sums of the Gaussian functions are shown as red dashed lines.