Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry

Abstract

Hydroxyl radical protein footprinting (HRPF) is a powerful method for measuring protein topography, allowing researchers to monitor events that alter the solvent accessible surface of a protein (e.g., ligand binding, aggregation, conformational changes, etc.) by measuring changes in the apparent rate of reaction of portions of the protein to hydroxyl radicals diffusing in solution. Fast Photochemical Oxidation of Proteins (FPOP) offers an ultrafast benchtop method for radical generation for HRPF, photolyzing hydrogen peroxide using a UV laser to generate high concentrations of hydroxyl radicals that are consumed on roughly a microsecond time scale. The broad reactivity of hydroxyl radicals means that almost anything added to the solution (e.g., ligands, buffers, excipients, etc.) will scavenge hydroxyl radicals, altering their half-life and changing the effective radical concentration experienced by the protein. Similarly, minute changes in peroxide concentration, laser fluence, and buffer composition can alter the effective radical concentration, making reproduction of data challenging. Here, we present a simple method for radical dosimetry that can be carried out as part of the FPOP workflow, allowing for measurement of effective radical concentration in real time. Additionally, by modulating the amount of radical generated, we demonstrate that effective hydroxyl radical yields in FPOP HRPF experiments carried out in buffers with widely differing levels of hydroxyl radical scavenging capacity can be compensated on the fly, yielding statistically indistinguishable results for the same conformer. This method represents a major step in transforming FPOP into a robust and reproducible technology capable of probing protein structure in a wide variety of contexts.

Figure 1.

Optical layout of the inline dosimeter. A V-groove positions a 362 μm OD capillary precisely at the optimal focal plane for the UV LED. Inset: Modeled light path for the UV LED. The vast majority of the light passes through the lumen of the capillary into the detector.

Figure 2.

Change in adenine absorbance as a function of hydrogen peroxide concentration. As the amount of hydroxyl radical generated increases, the dosimeter response increases linearly.

Figure 3.

Change in adenine absorbance as a function of radical scavenger concentration. As the amount of MES radical scavenger increases, the effective radical dose decreases and the dosimeter response decreases linearly.

Figure 4.. Correlation of adenine dosimetry and model peptide oxidation.

Oxidation of the GluB peptide varies linearly with inline adenine dosimetry at (A.) different laser fluences, 100 mM hydrogen peroxide; and (B.) 13.06 mJ/mm² fluence, different hydrogen peroxide concentrations. All error bars represent one standard deviation from a triplicate measurement.

Figure 5.

Real time compensation of myoglobin oxidation by inline adenine dosimetry. (Blue) Myoglobin oxidized in 10 mM phosphate buffer at 11.66 mJ/mm² yielded a ΔAbs_265nm of 19.13 ± 1.03 mAU. (Orange) Myoglobin oxidized in 10 mM MES buffer at 11.66 mJ/mm², yielding a reduced inline adenine dosimetry reading (ΔAbs_265nm of 7.07 ± 1.10 mAU). All peptides identified as oxidized gave a reduced amount of oxidation compared to the phosphate buffer myoglobin sample. (Grey) Myoglobin oxidized in 10 mM MES buffer at a higher laser fluence (18.75 mJ/mm²) using the adenine dosimetry response (ΔAbs_265nm of 18.17 ± 0.26 mAU) to compensate for differential scavenging between that of the 10 mM phosphate buffer sample. No peptide yielded statistically significant differences in oxidation between the compensated MES buffer sample and the phosphate buffer sample (α = 0.05).