Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

Abstract

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area of proteins. This technique relies on the reaction of amino acid side chains with hydroxyl radicals freely diffusing in solution. FPOP generates these radicals in situ by laser photolysis of hydrogen peroxide, creating a burst of hydroxyl radicals that is depleted on the order of a microsecond. When these hydroxyl radicals react with a solvent-accessible amino acid side chain, the reaction products exhibit a mass shift that can be measured and quantified by mass spectrometry. Since the rate of reaction of an amino acid depends in part on the average solvent accessible surface of that amino acid, measured changes in the amount of oxidation of a given region of a protein can be directly correlated to changes in the solvent accessibility of that region between different conformations (e.g., ligand-bound versus ligand-free, monomer vs. aggregate, etc.) FPOP has been applied in a number of problems in biology, including protein-protein interactions, protein conformational changes, and protein-ligand binding. As the available concentration of hydroxyl radicals varies based on many experimental conditions in the FPOP experiment, it is important to monitor the effective radical dose to which the protein analyte is exposed. This monitoring is efficiently achieved by incorporating an inline dosimeter to measure the signal from the FPOP reaction, with laser fluence adjusted in real-time to achieve the desired amount of oxidation. With this compensation, changes in protein topography reflecting conformational changes, ligand-binding surfaces, and/or protein-protein interaction interfaces can be determined in heterogeneous samples using relatively low sample amounts.

Figure 1:. Overview of FPOP.

The surface of the protein is covalently modified by highly reactive hydroxyl radicals. The hydroxyl radicals will react with amino acid side chains of the protein at a rate that is strongly influenced by the solvent accessibility of the side chain. Topographical changes (for example, due to the binding of a ligand as shown above) will protect amino acids in the region of interaction from reacting with hydroxyl radicals, resulting in a decrease in the intensity of modified peptide in the LC-MS signal.

Figure 2:. Kinetic simulation of dosimetry-based compensation using Tenua 2.0.

1 mM adenine dosimeter response is measured in 5 μM lysozyme analyte with a 1 mM initial hydroxyl radical concentration (▪OH t_1/2=53 ns), and set as a target dosimeter response (black). Upon the addition of 1 mM of the scavenger excipient histidine, the dosimeter response (blue) decreases along with the amount of protein oxidation in a proportional manner (cyan). The half-life of the hydroxyl radical also decreases (▪OH t_1/2=39 ns). When the amount of hydroxyl radical generated is increased to give an equivalent yield of oxidized dosimeter in the sample with 1 mM histidine scavenger as achieved with 1 mM hydroxyl radical in the absence of scavenger (red), the amount of protein oxidation that occurs similarly becomes identical (magenta), while the hydroxyl radical half-life decreases even further (▪OH t_1/2=29 ns). Adapted with permission from Sharp J.S., Am Pharmaceut Rev 22, 50-55, 2019.

Figure 3:. Optical bench for the FPOP experiment.

(A) The sample is mixed with H₂O₂, adenine radical dosimeter, and glutamine scavenger and loaded into the syringe. The sample is pushed through the fused silica capillary through the focused beam path of a KrF excimer UV laser. The UV light photolyzes H₂O₂ into hydroxyl radicals, which oxidizes the protein and adenine dosimeter. The syringe flow pushes the illuminated sample out of the path of the laser before the next laser pulse, with an unilluminated exclusion volume between illuminated regions. Immediately after oxidation, the sample is passed through an inline UV spectrophotometer, which measures the UV absorbance of adenine at 265 nm. The sample is then deposited into a quench buffer to eliminate the remaining H₂O₂ and secondary oxidants. (B) The spot size is measured after irradiating a colored Post-It note affixed behind the capillary with the laser at 248 nm. The width of the spot is used for calculating the sample flow rate, and the silhouette of the capillary in the center of the spot is used to align the optical bench.

Figure 4:. Extracted ion chromatogram of a peptide and its oxidation products after FPOP.

The m/z of the peptide oxidation products are calculated based on the m/z of the unoxidized peptide and the known oxidation products; and the areas of these peptide products are determined. The area of the peptide products is then used for the calculation of the average oxidation events per peptide.

Figure 5:. Peptide level footprint of the heavy chain of adalimumab.

The peptide average oxidation of adalimumab at room temperature (blue) and after adalimumab is heated at 55 ⁰C for 1 hr, then cooled to room temperature. The error bars represent one standard deviation of triplicate measurements. The asterisk represents the peptides that are significantly changed in the two conditions (p ≤ 0.05).

Figure 6:. Compensation of adenine dosimetry readings.

The adenine reading before and after laser irradiation were recorded for FPOP in phosphate buffer at 265 nm with using inline dosimeter. As MES is a good scavenger of the hydroxyl radicals, the difference in adenine readings was lower. Increased the laser fluence of FPOP solution with MES buffer to “compensate” and overcome the effect of MES buffer to have similar adenine reading as phosphate buffer.

Figure 7:. Real-time compensation of myoglobin oxidation by inline adenine dosimetry.

Myoglobin oxidized in 10 mM phosphate buffer (blue) and 10 mM MES buffer (orange). As noted, the oxidation of the peptides is lower in the MES buffer. As the laser fluence is increased for the samples in the MES buffer to have almost similar adenine dosimetry level as compared to phosphate buffer (grey), the peptide level oxidation is also similar to the oxidation level seen in samples with phosphate buffer. This figure has been adapted with permission from Anal Chem 2018, 90, 21, 12625-12630. Copyright 2018 American Chemical Society.