Hydroxyl Radical Dosimetry for High Flux Hydroxyl Radical Protein Footprinting Applications Using a Simple Optical Detection Method

Abstract

Hydroxyl radical protein footprinting (HRPF) by fast photochemical oxidation of proteins (FPOP) is a powerful benchtop tool used to probe protein structure, interactions, and conformational changes in solution. However, the reproducibility of all HRPF techniques is limited by the ability to deliver a defined concentration of hydroxyl radicals to the protein. This ability is impacted by both the amount of radical generated and the presence of radical scavengers in solution. In order to compare HRPF data from sample to sample, a hydroxyl radical dosimeter is needed that can measure the effective concentration of radical that is delivered to the protein, after accounting for both differences in hydroxyl radical generation and nonanalyte radical consumption. Here, we test three radical dosimeters (Alexa Fluor 488, terepthalic acid, and adenine) for their ability to quantitatively measure the effective radical dose under the high radical concentration conditions of FPOP. Adenine has a quantitative relationship between UV spectrophotometric response, effective hydroxyl radical dose delivered, and peptide and protein oxidation levels over the range of radical concentrations typically encountered in FPOP. The simplicity of an adenine-based dosimeter allows for convenient and flexible incorporation into FPOP applications, and the ability to accurately measure the delivered radical dose will enable reproducible and reliable FPOP across a variety of platforms and applications.

Figure 1. Dose response curve of adenine in condition of different concentrations of variables

UV absorbance at 260 nm of 1mM adenine, 20 μM ubiquitin, 17 mM glutamine with: (a) different concentrations of hydrogen peroxide (10, 20, 40, 80, 100 mM) in 50 mM sodium phosphate buffer; (b) 100 mM hydrogen peroxide in different concentrations of tris-HCl buffer (10, 20, 40, 80, 160 mM); (c) 100 mM hydrogen peroxide photolyzed in 50 mM sodium phosphate buffer using different photolysis laser power (10.5, 32.4, 48.3, 62, 70.7 mJ/pulse). Each data point represents the average of three FPOP oxidation replicates, with error bars representing one standard deviation.

Figure 2. Relationship between angiotensin oxidation and adenine dosimetry

(a) Standard angiotensin Sample with addition of adenine photolyzed in varying concentration of Hydrogen Peroxide. Squares, left y-axis - The centroid mass of oxidized and unoxidized angiotensin, 2+ charge state, with varying concentrations of hydrogen peroxide (10, 20, 40, 80, 100 mM); Triangles, right y-axis - Dose response curve between hydrogen peroxide concentrations and adenine UV reading. (b) Standard Angiotensin Sample with addition of adenine photolyzed in varying concentration of tris-HCl buffer. Square marker - The centroid mass of oxidized and unoxidized angtension, 2+ charge state, with varying concentrations of tris-HCl buffer (10, 20, 40, 80, 160 mM); Triangle marker - Dose response curve between tris-HCl buffer concentrations and adenine UV reading.

Figure 3

Plots of fraction oxidized per residue versus the change in absorbance for adenine for (a–c) 18 moderately reactive amino acids found to be oxidized by FPOP experiments; (d) five highly reactive amino acids found to be oxidized by FPOP experiments.