In Vivo Application of TurboID-based Proximity Labeling in Drosophila melanogaster

In this study, TurboID-based proximity labeling is used to map the interactions of zucchini proteins in the germline cells of drosophila ovary. Both well known and novel interactors of zucchini were identified. Notably, several novel interactors were found to be involved in the protein folding, membrane organization, and physical trafficking. In this study, TurboID is used for effective labeling in animal tissue while controlling background biotinylation. Labeling specificity and protein coverage are enhanced by optimizing controls and enriching biotinylated peptides post trypsinization. This approach enables systemic screening of protein interactions to uncover the functions and molecular mechanisms of proteins of interest.

[Narrator] To begin, apply a lab spoonful of smooth yeast paste prepared by dissolving yeast powder in triple distilled water to the wall of a vial. Take another vial containing newly hatched flies and gently tap it to collect the flies at the bottom. Invert this vial onto the vial containing the yeast paste and align the rims to prevent flies from escaping. Tap both vials together to transfer the flies to the bottom part of the vial containing the paste. Once the transfer is complete, place cotton caps securely on both vials and wait for three days. On the third day, prepare 100 micromolar biotin working solution by diluting one molar biotin stock in triple distilled water. Add 500 microliters of 0.5% proponic acid to the solution. Mix the biotin solution with dry yeast powder and stir into a paste. Add a lab spoonful of biotin yeast paste to the wall of a biotin food vial. Divide the flies into two groups. Transfer one group into a vial containing normal food as a control. Transfer the other group into a biotin food vial supplemented with biotin yeast paste to begin biotin elation. After 16 hours, transfer the flies into a new vial containing normal food. For drosophila ovary collection, pipette one milliliter of cold Grace's Insect Medium onto the dissection dish. Using fine tip forceps, place a female fly into the dissection dish and submerge it in the medium. Hold the thorax with one pair of forceps without crushing the body. Use another forceps to gently remove the tip of the posterior abdomen where the genitalia is located. Slowly squeeze the ovaries out using the forceps, starting from the upper abdomen and moving downward. Remove muscle nets and surrounding tissues without damaging the ovaries. Transfer clean ovaries into a 1.7 milliliter tube using forceps and keep the tube on ice. For the tissue lysis, add 500 microliters of 2% sodium dodecyl sulfate in trys buffered saline supplemented with a protease inhibitor cocktail into the tube containing the ovaries. Wait until the cells are lysed and the solution becomes sticky. Perform sonication using the conditions shown on screen. Next, for acetone precipitation, transfer the lysate into a five milliliter low binding tube. Add cold acetone into the tube at six times the sample volume. Vortex the tube briefly, then load it into a multi rotator and incubate overnight for approximately 16 hours at minus 20 degrees Celsius. Pellet the precipitated proteins by centrifuging at 15,000 G for 15 minutes at four degrees Celsius. Now, add two milliliters of a mixture solution composed of 90% cold acetone and 10% trys buffered saline. Mix the pellet and the solution by pipetting in an up and down motion. Vortex the tube briefly before loading it again into the multi rotator and incubating for two hours at minus 20 degrees Celsius. Pellet the precipitated proteins by centrifuging at 15,000 G for 15 minutes at four degrees Celsius. After discarding the supernatant, perform a spin at four degrees Celsius. Then, open the tube and allow the pellet to air dry for 10 minutes. Re-suspend the protein pellet in 500 microliters of eight molar urea. Transfer the solution into a one milliliter sized milli tube containing adaptive focused acoustics or AFA fiber. Sonicate the tube using the same parameters shown earlier before performing a bicinchoninic acid, or BCASA, to determine protein concentration. Denature the proteins using a thermo block set to 450 RPM for one hour at 37 degrees Celsius. Add five microliters of one molar dithiothreitol dissolved in triple distilled water to the tube to achieve a final concentration of 10 millimolar. Place the tube again on the thermo block for one hour at 37 degrees Celsius to reduce the proteins. Now add 55 microliters of 400 millimolar iodoacetamide solution into the sample tube to reach a final concentration of 40 millimolars before placing the tube back into the thermo block for alkylation. Dilute the sample by adding 50 millimolar ammonium bicarbonate solution until the total volume reaches four milliliters. Add four microliters of one molar calcium chloride stock solution into the tube to reach a final concentration of one millimolar. Pipette up and down to homogenize the solution. Next, add 150 microliters of one milligram per milliliter trypsin stock solution to the tube, maintaining a trypsin to sample ratio of one to 20. Digest the protein using a thermo block set to 450 RPM for 16 hours at 37 degrees Celsius. Wash 150 microliters of streptavidin conjugated magnetic beads per three milligrams of sample with one milliliter of two molar urea in trys buffered saline. Pipette one milliliter of the digested peptides into the tube containing beads and mix gently. Combine the mixture with the remaining peptide sample in a five milliliter low binding tube. After incubating for an hour, transfer one milliliter of the mixture into a 1.5 milliliter tube and place it on a magnetic rack for one minute. Wash the beads twice with one milliliter of two molar urea in 50 millimolar ammonium bicarbonate and once with one milliliter of triple distilled water before placing the tube back on the magnetic rack for one more minute and discarding the buffer. Next, pipette 100 microliters of the elucian buffer into the bead containing tube, mix gently, and elute peptides using a thermo block set to 300 RPM for five minutes at 60 degrees Celsius before placing the tube again on the magnetic rack. Now transfer the elute into a new 1.5 milliliter tube, avoiding any beads. The eluted sample is now ready to be dried and used for mass spectrometry analysis. This figure presents confocal imaging and proteomic interaction analysis of zucchini V5 TurboID to identify proximal and interacting proteins. Confocal images revealed strong overlapping expression between transgenes and proximal proteins following biotin feeding treatment as captured by V5 antibody and streptavidin conjugated Alexa Fluer 594 respectively. Gene ontology analysis of the zucchini V5 TurboID proteome revealed enriched biotinylated proteins involved in chaperone mediated protein folding, organization of the endomembrane system, and regulation of transport from the endoplasmic reticulum to the GGI apparatus.