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

Fast photochemical oxidation of proteins is an emerging technique for the structural characterization of proteins. Different solvent additives and ligands have varied hydroxyl radical scavenging properties. To compare the protein structure in different conditions, real-time compensation of hydroxyl radicals generated in the reaction is required to normalize reaction conditions.

The method facilitates measurement of protein confirmation and protein interactions. The buffer composition, sample purity and protein concentration requirements are flexible allowing the method to address challenging structural problems. This technique allows a compression of samples in a wide variety of different buffer systems.

Using our dosimetry method, researchers can monitor how well their labeling reactions are working in real time. One application of this technology is in drug development because it allows researchers to test widely different drug formulations and how they affect protein structure. To prepare the FPOP optical bench.

First turn on the laser and set the laser to external trigger, constant energy and no gas replacement. When the laser has warmed set the laser energy per pulse to between 80 to 120 milligrams per pulse and use a butane torch to gently burn the polyimide coding of the capillary at the spot at which the inline dosimeter will read the absorbent signal at 265 nanometers after laser exposure. Use a lint-free wipe and methanol to gently wipe the debris from the capillary and place the cleaned capillary through the beam path of the laser and into the inline dosimeter.

Press the lever on the top of the inline dosimeter to open the hinge and remove the magnetic holders. Place the capillary in the machine groove of the inline dosimeter using the magnetic holders to keep the capillary in place and close the dosimeter hinge over the capillary pressing the hinge until the lever locks in place. Click the start flash button in the dosimetry software to begin firing the excimer laser and set the preset repetition rate between 10 to 20 Hertz in the dosimetry software with the laser firing move the motorized stage through its range of motion, ensuring that the beam stays centered on the aperture and that the silhouette of the capillary can be observed throughout.

Then flush the capillary with water at 20 microliters per minute for at least one minute and click start data plus auto zero in the dosimeter software to zero the dosimeter to the water before beginning the data collection. To perform an FPOP experiment add two microliters of hydrogen peroxide to 18 microliters of FPOP solution and use a pipette to gently mix the solution quickly collect the solution at the bottom of the tube by centrifugation and immediately use a gas tight syringe to load the solution into a syringe pump. Click start pump and start data in the dosimeter software to start the flow on the syringe pump at the appropriate flow rate and use the inline dosimeter to monitor the real-time adenine reading until the absorbent signal at 265 nanometers stabilizes collecting the sample in a waste container.

To start firing the laser at the preset repetition rate end energy click the start flash button in the dosimeter software and use the inline dosimeter to monitor the real-time adenine reading. The difference in the absorbance at 265 nanometers with the laser off and the laser on will be the delta absorbents at 265 nanometers reading. To ensure that comparable hydroxyl radicals are available to react with proteins across different samples.

Compare the delta absorbents at 265 nanometers reading obtained with the inline dosimeter with the desire delta absorbents at 265 nanometers reading obtained from previous experiments or controls. If the reading is at the desired level collect the sample immediately after laser or radiation in the quench buffer. To compensate the effective radical dose to equalize the absorbance readings change the hydrogen peroxide concentration, change the laser energy per pulse or change the focal plane of the focusing lens to increase the laser fluence.

To make a greater than 10 milli absorbance unit change in the delta absorbents reading remake the sample with more or less hydrogen peroxide and rerun the sample as demonstrated. To make a small change in the absorbance reading in real time, use the 50 millimeter motorized stage to adjust the position of the focusing lens to adjust the focal plane of the incident beam. To measure the effective amount of hydroxyl radical present in the sample after laser radiation monitor the adenine delta absorbents at 265 nanometers in real time with an inline UV capillary detector using the motorized stage to adjust the lens position until the delta absorbents at 265 nanometers reading is equal to the desired reading.

Comparison of the heavy chain peptide footprint of the adilumumab biosimilar in phosphate buffer and after heating at 55 degrees Celsius for one hour reveals that the indicated peptide regions demonstrate significant protection from solvents when the protein is heated to form aggregates. Using an inline dosimeter in real time to monitor the difference in adenine absorbents before and after laser or radiation indicates that a comparable change in the adenine absorbents level is observed in MES buffer compared to phosphate buffer. While the average oxidation of the peptides is lower in the presence of MES buffer compared to phosphate buffer.

However, as the laser fluence is increased to have an equal adenine dosimetry response the average peptide oxidation values are almost the same after FPOP and MES buffer and phosphate buffer. As the dosimetry reading is affected by the laser fluence peroxide concentration as well as the radical scavenger. Take care that the dosimetry response between the sample survey close to each other.

Site directed mutagenesis is often used to probe sites protected in FPOP. This allows researchers to determine if changes are due to Alistair. This technique facilitates the comparison of proteins in different buffers or formulations allowing researchers to determine the effect of different additives on protein confirmation.