Laser-free hydroxyl radical protein footprinting makes identifying protein interaction sites and regions of confirmational change much easier, accelerating research in protein aggregation, structure, and stability studies. With inline radical dosimetry, users can adjust the effective hydroxyl radical load in real time, and with real time radical dosimetry, you can save experimental time and precious sample while improving labeling reproducibility. To begin, cleave a 250 micrometer inner diameter silica capillary to 27 inches with a silica cleaving stone, checking the ends of the capillary for a clean and straight cut.
Create two windows of roughly 15 millimeter length. From the lower end of the capillary, burn away the polyimide coating 90 millimeters up to create the photolysis window and 225 millimeters for the dosimeter window. Insert the lower end of the capillary just beyond the conical end of a nut and ferrule.
Add the capillary to port five, tightening just beyond finger tight with a wrench. Remove the photolysis cell cap of the photolysis module by pulling it straight out, then remove the magnetically mounted metal mask, which will hold the capillary in place. Open the dosimeter cell by pushing on the tab on the left and swing the dosimeter cell open to the right.
Remove the magnetically mounted clips that will hold the capillary in place. Then place the capillary into the grooved channel at the base of the photolysis cell, and center the capillary window with the photolysis cell window. Hold the capillary in position and add the magnetic mask.
Place the photolysis cap back in position. Place the capillary into the grooved channel at the base of the dosimeter cell, and center the second capillary window on the small aperture at the center of the dosimeter cell. While holding the capillary in position with one hand, place the two magnetic clips in position to hold the capillary in place.
Close the dosimeter cell until it clicks closed. Finally, insert the capillary through the knurled knob atop the capillary lift of the product collector, extending the capillary to just above the bottom of the vial. Cut the desired length of Teflon tubing using a cutter and check the ends for a clean straight cut.
Insert one end of the new injection loop through one of the nuts, and place a new ferrule onto the end of the tube. Hold the nut and ferrule in place while inserting the tube in port six of the injection valve until it bottoms out. Tighten the nut with a wrench, then confirm that the nut and ferrule are securely in place.
Once both ends have a nut and fixed ferrule, loosely screw one end to port three and the other end to port six. Once in position, tighten both sides to finger tight, then a quarter turn past finger tight with a wrench. Turn on the laser-free HRPF system by starting the fluidics module, followed by the photolysis module, dosimeter module, product collector, and the system computer.
Launch the system software. Fully submerge the tubing into 10 milliliters of buffer from the valve position on the syringe pump. Direct the tubing from the waste position and the tubing from port two on the syringe port to an empty container to collect waste.
Place 1.5 milliliter microcentrifuge tubes at the position marked HN6 on the product collector carousel. Rinse out the injection loop five times by injecting 25 microliters of HPLC grade water with the valve set to the load position. Manually turn the injection valve to flush the rest of the system.
Select Process on the control software to begin flowing water until a droplet forms. Make a solution containing two millimolar adenine and 10 micromolar protein. Then make a quench solution using 0.3 milligrams per milliliter catalase and 35 millimolar methianine amide.
Aliquot 25 microliters of quench solution into a 200 microliter micro tube. Dilute hydrogen peroxide to 200 millimolar, keeping it on ice. Start the flash voltage at zero volts on the control software.
This zero volt control will determine any background oxidation on the protein of interest. Select Start Data AutoZero, followed by Process then Ready. Finally, turn the injection valve down to the load position.
Place the quench solution in position one on the product collector carousel, then change the product vial to one on the system control software. Immediately before injection, mix 12.5 microliters of the adenine and protein mixture with 12.5 microliters of hydrogen peroxide. Pipette the mixture up and down to mix, then quickly spin it down.
Inject 25 microliters of this solution using the injection port within 10 seconds of mixing. Switch the injection valve to the inject position and wait while the sample is being processed. Turn up the flash voltage to 750 volts on the control software, and repeat the labeling steps.
Then record the absorbance of each sample. First, click Select, then manually select the beginning and end of the peak absorbance. In the available space, write in a description of the sample.
Repeat this for all acquired data. Copy and paste the data into a spreadsheet to calculate the average change in adenine absorbance for each voltage. After all samples have been collected, flush out the syringe port and sample loop by setting the injection valve down to the load position and inject 25 microliters of HPLC water five times.
Turn the injection valve up to the inject position to flush the rest of the system with HPLC water. Once flushed, stop the flow, exit the system control software, and turn off the modules, starting with the product collector, dosimeter module, photolysis module, and finally the fluidics module. Upon oxidation, adenine used for dosimetry decreases in UV absorbance at 265 nanometers.
Buffer 2 contains a radical scavenger, which decreases the change in adenine absorbance compared to Buffer 1. Apomyoglobin was modified in the presence of hydrogen peroxide and adenine at four increasing plasma lamp voltages. Six peptides were detected, with a linear increase in oxidation with respect to the change of adenine absorbance at 265 nanometers.
These six oxidized peptides are labeled on a crystal structure of myoglobin. The extent of oxidation detected from the high pressure plasma lamp is much higher than the laser-based method. This increase arises from the high pressure plasma lamp's broad spectrum UV emission spectrum.
By producing a broad UV spectrum, the high pressure plasma lamp better overlaps the absorbance domain of hydrogen peroxide, resulting in a more efficient production of hydroxyl radicals through the photolysis of hydrogen peroxide, as compared to the Kr F Excimer Laser and Nd YAG Laser. The high pressure plasma lamp significantly increases the production of hydroxyl radicals beyond what is typically observed using a Kr F Excimer Laser. Using 100 millimolar peroxide can cause high levels of background oxidation.
If the protein system is susceptible to peroxide-induced oxidation, decrease the concentration to as low as 10 millimolar. HRPF's irreversible label allows for downstream sample handling, including long protease digestions, you can add in tandem mass tag for multiplexing, and perform 2D chromatography, all improving peptide detection and concoordinate structural information.
