In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo FPOP protein-protein interactions and protein structure changes in C-elegans. These animals are widely used as a model system to study human disease. By coupling in vivo FPOP to mass spectrometry, we can probe protein-protein interactions within different cellular and body compartments in a live animal without the need of isolating one particular protein.

Begin the flow system assembly, by cutting a two centimeter piece of fluorinated ethylene propylene or FPP tubing. Use a clean dissecting needle to widen the inner diameter at one end of the tubing, creating a small crater about 15 millimeters in length. To create the infusing lines of the flow system, cut to 15 centimeter pieces of 250 micrometer inner diameter fused silica with a ceramic cutter.

And tape the two capillaries together with self adhesive tape, making sure that the ends are 100%flushed. Insert the two capillaries into the crater of the FPP tubing, pushing them up to the very edge. Place a small dot of epoxy resin on a clean surface, and mix it with the dissecting needle.

Use the same needle to quickly place a small drop of the resin, at the end of the infusing capillaries, where they connect with the FPP tubing, allow the resin to dry outlet side up for a few minutes. Meanwhile, cut a new 250 micrometer inner diameter capillary which will become the outlet capillary of the flow system. Once the resin has dried, insert the new capillary to the FPP tubing outlet end.

The inside ends of the outlet capillary and the two infusing capillaries should be flush against each other, creating the mixing tea. Bind to the capillary and FPP tubing with fresh epoxy resin as previously described, and allow the flow system to dry overnight. Insert four magnetic stirrers, inside one five milliliter syringe which will prevent worms from settling in this syringe during in vivo fast photochemical oxidation of proteins or in vivo FPOP.

Fill this and an additional five milliliter syringe with M9, making sure to avoid creating bubbles. Connect a lower adapter to each syringe, making sure that they are finger tight and secured in place. Then, attach each syringe to the middle port of a single three-two valve.

Secure each syringe to the dual syringe pump, and adjust the mechanical color to prevent overpressure from the pusher block. Use a super flange list not FEP sleeve and super flange list variable to attach each infusing capillary end of the previously made microfluidic system to the top port of each three-two valve. Finally, attach a 10 centimeter 450 micrometer inner diameter capillary to the bottom port of the valve, which will serve as the withdrawing sample capillary.

Start the pump flow and visually inspect all connections for leaks. Flow at least three syringe volumes using the experimental flow rate. The flow path is marked by the arrows on the three-two valve handle, and each syringe can be refilled manually by moving the valve handle from expelling to withdrawing position.

After inspecting the microfluidic flow system, move it to the experimental bench, and secure the outlet capillary to the radiating stage with a stainless steel union. Use a long reaching lighter to burn the coating of the fused silica at the laser irradiation window and clean the burned coating with lint tissue and methanol. Position the magnetic stirrer block above the syringe with the magnetic stirs and adjust the speed, so that the stirs are rotating slowly and constantly.

Turn on the Krypton fluoride excimer laser and allow the thyroid tron to warm up. Measure the laser energy at a frequency of 50 hertz for at least 100 pulses by placing the optical sensor at the beam exit window. Manually withdraw approximately 10, 000 worms in a 500 microliter volume into the sample syringe.

Then fill it with 2.5 milliliters of the M9 buffer, making sure to avoid air bubbles. It is important to have a sample size of at least 10, 000 worms. A smaller sample size will result in a low protein yield for downstream proteomic analysis.

Fill the second syringe with three milliliters of 200 millimolar hydrogen peroxide. At the end of the outlet capillary, place a 15 milliliter conical tube with six milliliters of 40 millimolar DMTU, 40 millimolar PBN, and 1%time applesauce oxide. Start the excimer laser from the software window, wait for the first pulse and start the sample flow from the dual syringe.

Collect the entire sample in the 15 milliliter tube, while actively monitoring the sample flow for any visual leaks. After in vivo FPOP, pellet the worms by centrifugation at 805 times g for two minutes. Aspirate the quench solution and add 250 microliters of licensed buffer.

Transfer the sample to a clean micro centrifuge tube, flash freeze it and store at negative 80 degrees Celsius until sample digestion. worm recovery was compared after in vivo FPOP using capillaries with two different internal diameters. When a 250 micrometer inner diameter capillary was used, a total sample recovery between 63 and 89%was achieved across two biological replicates.

Using a 150 micrometer inner diameter capillary resulted in only 21 to 31%recovery. The use of a larger inner diameter capillary, leads to better worm flow during in vivo FPOP, the smaller capillary does not allow for single worm flow, and multiple worms are seen flowing together at the laser radiating window, which decreases the amount of laser exposure per worm. Representative extracted ion chromatograms of an FPOP modified and unmodified peptide showed that the hydroxyl radical changes the chemistry of oxidatively modified peptides making them more polar.

In reverse phase chromatography, in vivo FPOP modified peptides have earlier retention times than unmodified peptides. MS fragmentation of isolated peptides, allows for the identification of oxidatively modified residues. In vivo FPOP has oxidatively modified a total of 545 proteins across two biological replicates within C-elegans.

One advantage of this protein footprinting method, is its ability to modify proteins, in a variety of body systems within the worms. Tandem MS analysis confirms in vivo FPOP probe solvent accessibility in vivo. The oxidation pattern of the heat shock protein 90 in complex with the myosin chaperone protein UNC 45, was analyzed and four oxidatively modified residues, were found.

The use of C-elegans for in vivo FPOP, offers the potential to study the role of protein-protein interactions and protein structure in disease pathogenesis.