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Method Article

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

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DOI:

10.3791/68193

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June 13th, 2025

 , 
1Soonchunhyang Institute of Medi-bio-Science, Soonchunhyang University, 2Department of Integrated Biomedical Science, Soonchunhyang University

In This Article

Summary

In this study, we establish a detailed protocol for TurboID-based proximity labeling (PL) in D. melanogaster ovary, covering steps from biotin supplementation and ovary dissection to transgene expression validation and enrichment of biotinylated peptides for mass spectrometry.

Abstract

Proximity labeling (PL) is a powerful method for mapping protein-protein interaction networks. Advances in labeling enzymes, especially TurboID, have broadened its use across biological systems. TurboID is particularly advantageous for in vivo applications due to its robustness, non-toxicity, and ease of use. This enzyme promiscuously biotinylates nearby biomolecules, including proteins and nucleic acids, within an approximately 10 nm range in the presence of biotin. We applied this method to investigate the interactome of Zucchini (Zuc), a key factor in piwi-interacting RNA (piRNA) biogenesis on the outer mitochondrial membrane (OMM) in germline cells of the Drosophila melanogaster ovary. By expressing Zuc-TurboID in germ cells and supplementing with biotin, we induced the biotinylation of Zuc-proximal proteins. Mass spectrometry analysis following the enrichment of biotinylated peptides identified proteins in close proximity to Zuc, including well-known interactors in piRNA biogenesis. Interestingly, we discovered novel interactors of Zuc involved in protein folding, membrane organization, and vesicle trafficking.

Introduction

Conventional protein-protein interaction (PPI) mapping methods, such as affinity purification-mass spectrometry (AP-MS) and yeast-2-hybrid (Y2H) systems, often fail to capture low-abundance, transiently expressed, or membrane-bound proteins. This limitation arises from their inability to replicate the native physiological conditions of a living cell1,2,3. To overcome these challenges, PL has emerged as a powerful technique for high-resolution proteome mapping at a sub-organelle level in vivo. PL leverages the fusion of a promiscuous enzyme with bait protein, enabling the enzyme to catalyze the formation of short-lived reactive molecules1. These reactive molecules, such as biotin, covalently label proteins within a few nanometers of the fusion protein4. Subsequently, biotinylated proteins are isolated and analyzed by mass spectrometry to facilitate large-scale protein identification5,6.

Over the past years, various promiscuous enzymes have been developed to advance PL techniques. Two widely used enzymes are Escherichia coli-derived R118G mutant biotin ligase (BioID) and pea-derived ascorbate peroxidase enzyme 2 (APEX2). BioID converts biotin and ATP into biotinyl-5'-adenylate (bioAMP)1. This reactive molecule covalently binds to lysine residues of nearby proteins within ± 10 nm radius. A key advantage of BioID-based PL is the use of biotin. It is a naturally occurring and non-toxic biomolecule in living organisms, which allows for safe in vivo labeling6. However, BioID has limitations, including slow labeling kinetics (18-24 h) and the requirement for high temperature (37 °C) for optimal catalytic activity4. These traits can hinder its application in studying dynamic biological processes. Meanwhile, APEX2 catalyzes the formation of biotin-phenoxyl radicals in the presence of biotin-phenol and hydrogen peroxide (H2O2)7. These radicals mainly react with the side chain of electron-rich amino acids like tyrosine and can also bind cysteine, histidine, and tryptophan8. The rapid labeling capability of the peroxidase enzyme (< 1 min) is suitable for capturing dynamic and transient PPIs9. Moreover, it has a wider detection range (up to 20 nm) than biotin ligase. However, APEX2-based PL has its drawbacks, including the requirement for a toxic oxidizing agent, H2O2, and the low permeability of biotin-phenol, which limit the applicability in vivo10.

In 2018, Branon et al. developed a new biotin ligase called TurboID, a 35 kD mutant version of BirA (the biotin ligase found in E. coli)4. This enzyme incorporates a mutation at the R118 position (R118S) together with 15 other mutations relative to BirA. It exhibits two-fold higher catalytic activity than BioID. TurboID-induced biotinylation in 10 min produces proteomic data with a size and specificity comparable to 18 h of labeling by BioID. Furthermore, it demonstrates excellent labeling activity at 30 °C4. Therefore, TurboID is more suitable for application in organisms like Drosophila or Caenorhabditis elegans, which are commonly reared at 25 °C and 20 °C, respectively.

In this study, we aim to utilize TurboID-based PL for interactome study in the Drosophila ovary. The protocols described here are employed to map the interactome of Zuc in germline cells. Zuc is an endonuclease localized on the outer mitochondrial membrane (OMM), which mediates the biogenesis of piRNAs, small non-coding RNAs critical for the maintenance of genome integrity11,12,13. By comparing biotinylated proteins identified from Zuc-TurboID analysis with those from two additional controls, NES-TurboID and Tom20-TurboID, we defined distinct Zuc-interacting candidates.

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Protocol

1. PL in adult fly

  1. Preparation of biotin food
    1. Prepare 1 M NaOH solution by diluting 10 M NaOH stock in triple distilled water (TDW). Store it at room temperature (RT). Prepare 1 M biotin stock solution by dissolving 0.0049 g biotin powder in 20 mL of TDW. Add 150-200 µL of 1 M NaOH to increase biotin solubility. Vortex to homogenize the solution. Store biotin stock solution at 4 °C.
    2. Take out fly food from the vial and microwave it for 2 min until it is melted.
      NOTE: Standard fly food contains 5% cornmeal, 3% yeast, 10% sucrose, 1% agar, 0.3% propionic acid, and 0.3% methyl 4-hydroxybenzoate13.
    3. Add biotin stock to the melted food with a final concentration of 100 µM, then mix by vortexing. Immediately dispense 4.5 mL of biotin-supplemented food into each empty vial using a serological pipette. Let the food cool down for 30 min, place a cotton cap on the vial, then store the biotin food at 4 °C.
      NOTE: Biotin must be added before the food cools down and hardens.
  2. Biotin feeding
    1. Cross the transgenic flies that overexpress TurboID fusion protein with tissue or cell-specific GAL4 flies14,15,16. As a negative control, cross wild-type flies with GAL4 driver flies to ensure no biotinylation induction.
    2. Around 10 days after crossing, progenies will start to hatch. Dissolve dry yeast powder into TDW and stir into a smooth paste. Apply a lab spoonful of yeast paste to the wall of a new vial. Gently tap down the old vial containing newly hatched flies to collect the flies at the bottom. Invert the old vial onto the new vial containing yeast paste, ensuring the rims of both vials are aligned to prevent flies from escaping.
    3. Tap down those vials together to transfer flies from the old vial to the bottom part of the new vial. Once the flies have been transferred, securely place cotton caps back on both the new and old vials. Wait for 3 days. Yeast paste can be stored at RT for a few days.
      NOTE: Fly transfer needs to be done as quickly as possible because flies will only be stunned temporarily.
    4. Prepare 0.5% propionic acid solution by diluting 250 µL of propionic acid stock in 50 mL TDW. Store it at RT.
    5. On the 3rd day, prepare 100 µM biotin working solution by diluting 1 M biotin stock in TDW, then add 500 µL of 0.5% propionic acid. Mix biotin working solution with dry yeast powder and stir until it forms a paste. Add a lab spoonful of biotin-yeast paste to the wall of the biotin food vial. The biotin-yeast paste can be stored at RT for a few days.
    6. Divide flies into two groups. Keep one group in the normal food vial as a control and transfer the other group to the biotin food vial (biotin-yeast paste added) to start biotinylation. After 16 h of biotinylation, transfer flies into a new normal food vial.
      NOTE: We previously performed a biotin feeding test for 4 h, 8 h, and 16 h. The biotinylation signal was strongest after 16 h feeding, so we chose this time point for subsequential experiments.
  3. Drosophila ovary collection
    1. Put flies into the CO2 fly pad, keep the females, and discard the males. After the flies are properly anesthetized (not moving anymore), pipette 1 mL of cold Grace's insect medium onto the dissection dish.
    2. Take a fly to the dissection dish using fine-tip forceps and let it submerge in the medium. Hold the fly thorax with forceps, be careful not to crush the body. Then, use another forceps to gently remove the tip of the posterior abdomen (where the genitalia part is located).
    3. Use the forceps to slowly squeeze the ovaries out (starting from the upper abdomen down to the bottom).
    4. Once they are out, remove muscle nets and other tissues that surround the ovaries. Be careful not to damage the ovaries.
    5. Carefully transfer clean ovaries into a 1.7 mL tube using forceps; keep the tube on ice. Repeat steps 1.3.2 - 1.3.4 for the next flies.
    6. Spin down the tube at 3,500 x g for 30 s at 4 °C, then use micropipette tips to remove the remaining medium. Be careful not to touch the ovaries.
    7. Snap-freeze the tube with liquid nitrogen, then store the ovaries at -80 °C. The ovary samples are stable for 1 year.

2. Transgene expression and biotinylated protein detection by Western blot assay

NOTE: For western blot assay, collect around five ovary pairs per sample.

  1. Tissue lysis
    1. Thaw the frozen ovary tissue. Add 50 µL of RIPA lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100), supplemented with 1x protease inhibitor (PI) cocktail and 1 mM Phenylmethanesulfonyl Fluoride (PMSF). Store RIPA lysis buffer (without 1x PI cocktail and 1 mM PMSF) at 4 °C.
      NOTE: PI cocktail and PMSF should only be added right before the experiment.
    2. Disrupt ovaries using a hand-held homogenizer coupled with a plastic pestle (1 min per tube). A tissue lysis process on ice for 10 min is necessary to ensure complete lysis, followed by homogenization.
    3. Centrifuge at 13,500 x g for 10 min at 4 °C to separate protein lysate from debris.
    4. Pipette clear protein lysate to a new 1.7 mL tube (avoid taking the lipid layer on top), then store lysate at -20 °C until further use.
  2. Protein concentration measurement by Bicinchoninic acid (BCA) assay
    1. Prepare Bicinchoninic acid (BCA) working reagent (WR) by mixing reagent A and reagent B in a 50:1 ratio. Mix 1 µL of lysate or diluted BSA standard with 200 µL of WR in a 96-well plate (in duplicates), then incubate the plate at 37 °C for 30 min.
      1. Calculate how much WR is necessary using this formula:
        (#  standards + # samples) × (# replicates) × (WR volume per sample)
        ​To make a set of protein standards, prepare several dilutions of 2 mg/mL bovine serum albumin (BSA) in RIPA lysis buffer (without PI cocktail and PMSF) with final concentrations 0, 25, 125, 250, 500, 750, 1000, 1500, and 2000 µg/mL.
    2. Measure absorbance using a microplate reader at 495 nm wavelength. Generate a standard curve from BSA absorbance values, then determine protein concentration.
  3. Western blot assay
    1. Gel electrophoresis
      1. Add 30-50 µg protein lysate to the 2x SDS loading buffer. Add TDW (if necessary) to make a final 2 µg/µL protein concentration and 1x SDS final concentration, then boil the sample at 95 °C for 10 min.
      2. Prepare running buffer by mixing 25 mL of 20x MOPS buffer with 500 mL of TDW to make 1x concentration of running buffer. Store it at RT.
      3. Rinse the wells of Bis-Tris gel with 1 mL of running buffer (3x), then place Bis-Tris gel into the electrophoresis chamber. Fill the front side of the chamber with the new running buffer until all the wells are submerged (± 250 mL). Fill the back side of the chamber with the used running buffer (half the volume of the new running buffer used is sufficient).
      4. Load the sample onto Bis-Tris gel and use 4 µL of prestained protein ladder as the marker. Perform electrophoresis (200 V, 30 min). Remove the gel from the cassette and rinse with TDW.
    2. Membrane blotting
      1. Make transfer buffer by adding 20 mL of transfer buffer (25x stock) and 50 mL of methanol into 430 mL of TDW.
      2. Arrange a sandwich consisting of blotting pads, filter paper, gel, and nitrocellulose membrane in a blot module. Before stacking a new layer, use a roller to remove bubbles.
      3. Install the blot module onto the transfer chamber, then fill the chamber with 500 mL of transfer buffer. Transfer proteins for 1 h at 175 mA.
        NOTE: Transfer buffer should be freshly prepared before the experiment.
    3. Blocking
      1. Add 5 mL of Tween20 stock into 45 mL of TDW to make 10% Tween20 stock solution. Dissolve two phosphate-buffered saline (PBS) tablets in 400 mL of TDW. Remove 4 mL of solution, then add 4 mL of 10% Tween20 (final concentration 0.1%) to make 1x PBST solution. Store it at RT.
      2. Remove the blotted membrane from the transfer chamber, then rinse with 1x PBST.
        NOTE: If the membrane needs to be incubated with different antibodies, cut it with a clean stainless-steel blade according to expected protein sizes before performing the blocking step.
      3. To check gene expressions, use 2% BSA in 1x PBST for the blocking step (1 h, RT). Make 2% BSA solution by adding 1 g BSA into 50 mL of 1x PBST. For biotinylated protein detections, perform blocking using 3% BSA in 1x PBST (overnight, 4 °C). Dissolve 1.5 g BSA powder in 50 mL of 1x PBST to make a 3% BSA solution. Store blocking solutions at 4 °C.
        NOTE: The BSA used is different from the one used in the BCA assay. BSA for BCA assay is part of the BCA kit.
    4. Primary antibody incubation
      1. Dilute 1 µL of α-V5 in 10 mL of 2% BSA (1: 10,000) and 10 µL of housekeeping gene α-HSP90 in 2% BSA (1: 1,000). Add 2.7 µL of streptavidin-horseradish peroxidase conjugate (SA-HRP) into 9 mL of 1x PBST solution to make 0.3 µg/mL SA-HRP solution.
      2. To check gene expressions, incubate the membrane with primary antibody overnight at 4 °C. For biotinylated protein detections, incubate the membrane for 1 h at 4 °C. Store primary antibody back at 4 °C. It can be used multiple times.
      3. Wash the membrane with 1x PBST for 3x at RT for 15 min each.
    5. Secondary antibody incubation
      1. Prepare secondary antibody solution by adding 2 µL of either Goat anti-rabbit or anti-mouse IgG (horseradish peroxidase (HRP) conjugated) into 10 mL of 2% BSA (1: 5,000).
        NOTE: For SA-HRP, a secondary antibody is not needed. Proceed directly to the visualization step. Secondary antibodies should be freshly diluted before the experiment.
      2. Incubate membrane in secondary antibody solution for 40 min at RT. Wash membrane with 1x PBST for 3x at RT for 15 min each.
    6. Protein detection with enhanced chemiluminescence (ECL) method
      1. Mix 750 µL of luminol and 750 µL of HRP solution (1:1 ratio). Keep the membrane moist in the washing buffer while preparing the mixture.
        NOTE:This mixture should be freshly prepared before the experiment. As the chemicals are light-sensitive, keep them away from sunlight or other intense light sources (short-term exposure to lab lighting is fine).
      2. Dip the membrane in the solution for 30 s, then place it in between transparent plastic sheets. Observe blotted protein bands with a Chemidoc instrument.

3. Transgene expression and localization validation by immunofluorescence staining

NOTE:To check gene expression and localization, dissect around five ovary pairs per sample from 2-day-old females fed with yeast paste. Young ovaries predominantly contain early stages of egg chambers, which are ideal for immunofluorescence staining due to the abundance of nurse cells (germline cells) and their relatively permeable membranes17. Late stages of egg chambers are more rigid and difficult to penetrate due to the formation of the eggshell complex, which begins at stage 918. For the protein biotinylation test, dissect around five ovary pairs per sample from 4-day-old females fed with biotin, as described in step 1.2.

  1. Sample preparation
    1. Hold the posterior part of a dissected ovary in place using forceps. Use another forceps to gently tease apart the ovary into individual egg chambers. Work carefully to avoid rupturing the egg chambers. The egg chamber isolation protocol was described in detail in19. Carefully pipette egg chambers into a 1.7 mL tube, then wash with 1 mL of 1x PBS (RT). Cut the edge of the pipette tips before pipetting to prevent egg chambers from getting stuck in the tips.
      NOTE: Only use egg chambers at stage ≤ 9 (transparent ones). Discard mature oocytes and late-stage egg chambers ≥ 10. Pipetting will cause egg chambers to float in the solution. Therefore, let the egg chambers settle at the bottom (for 5 min) before proceeding to the next steps.
  2. Fixation
    1. Dilute 3.125 mL of 16% formaldehyde (FA) stock in 6.875 mL of 1x PBS to make a 5% FA solution. Store it at RT.
      NOTE: FA is light-sensitive, so keep it away from light.
    2. Pipette 1 mL of 5% Formaldehyde (FA) into the tube containing oocytes. Place the tube onto the multi-rotator, then fix the oocytes for 30 min.
      NOTE: From fixation until the nuclear-staining step, experiments are performed on a multi-rotator with gentle rotation.
    3. Prepare 0.5% Triton X-100 in 1x PBS (1x PBT) as a washing solution by adding 10 mL of 10% Triton X-100 stock into 190 mL of 1x PBS. Store it at RT.
    4. Rinse fixed egg chambers 1x, followed by washing 3x at RT for 10 min. Use 1 mL of 1x PBT for both rinsing and washing steps.
  3. Blocking
    1. Prepare 5% fetal bovine serum (FBS) by adding 250 µL of FBS stock into 4.75 mL of 1x PBT.
      NOTE: This solution should be freshly made before the experiment.
    2. Add 1 mL of 5% FBS into the tube and perform the blocking step for 1 h at RT. Rinse oocytes 1x.
  4. Primary antibody incubation
    1. For checking transgene expression and localization, dilute 2 µL of α-V5 and 2 µL of α-ATP5A or α-Tom20 (as a mitochondrial marker) in 400 µL of 1x PBT (1:200 ratio).
      NOTE: Although two antibodies can be used together to stain one sample, their hosts need to be different. It is to avoid overlapping binding of subsequent secondary antibodies.
    2. Incubate egg chambers with 400 µL of diluted primary antibody solution (overnight, 4 °C).
      NOTE: Similar to Western blot assay, primary antibodies can be used multiple times for immunostaining. Before storing an antibody solution back, ensure it does not contain any egg chambers.
    3. Rinse egg chambers 1x, followed by washing 3x at RT for 10 min.
  5. Secondary antibody incubation
    1. Dilute 2 µL of Goat α-Rb IgG Alexa Fluor 488 and 2 µL of Goat α-M IgG Alexa Fluor 594 in 400 µL of 1x PBT (1:200 ratio). Incubate egg chambers with 400 µL of diluted secondary antibody solution for 2 h at RT. For biotinylation detection, add 2 µL of streptavidin-conjugated Alexa Fluor 594 to 1x PBT (1:200 ratio) and incubate together with a secondary antibody.
      NOTE: From this step, protect the sample from light. Secondary antibodies should be freshly diluted before the experiment. Multiple secondary antibodies can be used together, but each primary antibody should be assigned to different fluorophores that originated from different hosts as well to avoid overlapping signals. For example, 488 Goat α-Rb will bind to rabbit α-V5. As for mouse α-ATP5A, 594 Goat α-M is used as its complementary secondary antibody. In the case of biotinylation detection, the use of other dyes with the same wavelength as streptavidin conjugate should be omitted.
    2. Rinse egg chambers 1x, followed by washing 3x at RT for 10 min.
  6. Nuclear staining
    1. Dilute 1 µL of DAPI in 1 mL of 1x PBS (1:1,000 ratio). Incubate egg chambers with 1 mL of diluted DAPI solution for 5 min at RT.
      NOTE: DAPI should be freshly diluted before the experiment.
    2. Rinse egg chambers 1x, followed by washing 3x at RT for 10 min.
      NOTE: Egg chambers can be stored in 1x PBS solution at 4 °C for a few hours before mounting, but it is recommended to proceed as soon as possible.
  7. Slide mounting
    1. Clean the glass slide with lint-free tissue, then write down sample information on it. Remove 1x PBS from the tube, but leave a small amount to prevent dryness.
    2. Cut the edge of micropipette tips, then carefully pipette egg chambers onto the glass slide. Use forceps to carefully distribute egg chambers on the glass slide and avoid any clumping. Observe the slides under a microscope (2x magnification is sufficient) to ensure that egg chambers are equally distributed.
    3. Remove the remaining PBS from the glass slide using a micropipette. Pipette 15-30 µL of antifade solution (light-sensitive) onto the glass slide and ensure it comes into contact with all egg chambers.
    4. Use forceps to hold the slide cover edge, then place it on top of the glass slide (starting from one edge to the other) so that it covers egg chambers. Do it slowly to avoid any bubbles. Blot excess solution with lint-free tissue, then seal the coverslip edges using nail polish. Let nail polish properly dry (± 5 min).
    5. Image the slides with a confocal microscope. Store slides at -20 °C.

4. Biotinylated peptide enrichment for mass spectrometry analysis

NOTE: Around 3 mg proteins are needed for the mass spectrometry sample. Therefore, dissect around 100 ovary pairs to obtain that amount.

  1. Tissue lysis
    1. Thaw the frozen ovary tissue. Meanwhile, prepare 1x Tris-buffered saline (TBS) solution by dissolving 1 TBS tablet in 500 mL of TDW. Store it at RT. Prepare 2% SDS in 1x TBS as lysis buffer by diluting 4 mL of 10% SDS stock in 16 mL of 1x TBS. Store it at RT.
    2. Add 500 µL of 2% SDS in 1x TBS (supplemented with 1x PI cocktail) into the tube containing ovaries. Wait for several minutes until cells are lysed and the solution turns sticky.
    3. Pipette lysate to 1 mL sized militube containing adaptive focused acoustics (AFA) fiber. Cut pipette tips before use. To ensure efficient sonication, transfer a maximum of 500 µL of lysate per tube.
    4. Perform sonication with the following conditions: peak incident power 75 W, duty factor 10%, cycles per burst 200, time 180 s, temperature 6 °C.
      NOTE: If a sample must be subjected to sonication multiple times, set up some intervals in between to avoid generating excess heat.
  2. Acetone precipitation
    1. Move lysate to a 5 mL low-binding tube, then add cold acetone into the tube at 6x of the sample volume. For example, add 3 mL of acetone to 500 µL of lysate.
      NOTE: Acetone should be pre-chilled at -20 °C before use.
    2. Vortex tube, load it into multi-rotator and incubate overnight (± 16 h) at -20 °C.
      NOTE: This is a stop point, which means the experiment can be momentarily paused after this step.
    3. Pellet precipitated proteins at 15,000 x g for 15 min at 4 °C. Discard supernatant, then spin down the tube at 4 °C.
    4. Add 2 mL of a mixture solution consisting of 90% cold acetone and 10% 1x TBS. Pipette in an up-and-down motion to mix the pellet and solution. Cut the pipette tips before use.
    5. Vortex tube, then load it into the multi-rotator and incubate for 2 h at -20 °C.
    6. Pellet proteins at 15,000 x g for 15 min at 4 °C. Discard the supernatant, then spin down the tube at 4 °C. Open the tube and air-dry the pellet for 10 min.
      NOTE: Do not over-dry the pellet because it will be difficult to resuspend later.
    7. Prepare 500 mM ammonium bicarbonate (ABC) stock solution by dissolving 0.7906 g ABC in 20 mL of TDW. Make 50 mM ABC working solution by diluting 3 mL of 500 mM ABC stock solution in 27 mL of TDW.
    8. Prepare 8 M urea solution by adding 0.4805 g urea in 1 mL of 50 mM ABC.
      NOTE: Urea solution should be freshly prepared before the experiment.
    9. Resuspend protein pellet in 500 µL of 8 M urea. Volume of 8 M urea can be reduced to increase protein concentration.
    10. Transfer resuspended protein into 1 mL sized militube containing AFA fiber, then perform sonication with the same conditions described in step 4.1.4.
    11. Perform BCA assay to determine protein concentration. This step is a stop point. Store the protein sample at 4 °C until further use.
  3. Trypsin digestion
    1. Mix 3 mg proteins with TDW (if necessary) in a 5 mL low-binding tube to make a total of 500 µL of solution.
    2. Denature proteins using thermoblock at 450 rpm for 1 h at 37 °C.
    3. Add 5 µL of 1 M DTT (dissolved in TDW) into the tube to make a final concentration of 10 mM DTT, then reduce proteins using a thermoblock at 450 rpm for 1 h at 37 °C.
    4. Prepare 400 mM iodoacetamide by dissolving 0.0111 g iodoacetamide in 150 µL of ABC. Add 55 µL of 400 mM iodoacetamide into the tube (final concentration 40 mM), then alkylate proteins using thermoblock at 450 rpm for 1 h at 37 °C.
      NOTE: Iodoacetamide is light-sensitive, and all iodoacetamide solutions should be freshly prepared before the experiment.
    5. Dilute the sample by adding 50 mM ABC solution till the total volume reaches 4 mL. After dilution, urea concentration in the solution is around 1 M.
    6. Make 1 M CaCl2 stock solution by dissolving 1.1098 g CaCl2 in 10 mL of TDW. Add 4 µL of 1 M CaCl2 stock into the tube to make a 1 mM final concentration, then pipette up and down to homogenize the solution.
    7. Prepare a 1 mg/mL trypsin stock solution by adding 0.001 g trypsin to 1 mL of ABC. Add 150 µL of trypsin to the tube (trypsin: sample = 1:20 ratio). Digest protein using thermoblock at 450 rpm for 16 h at 37 °C.
      NOTE: Trypsin should be freshly dissolved before the experiment. This step is a stop point. Store digested peptides at 4 °C until further use.
  4. Biotinylated peptide enrichment
    NOTE: Wash and elution buffers should be freshly prepared before the experiment.
    1. Prepare 2 M urea in 1x TBS solution by adding 1.2012 g urea in 10 mL of 1x TBS.
    2. Use 150 µL of streptavidin-conjugated magnetic beads per 3 mg sample. Wash the streptavidin beads with 1 mL of 2 M urea in 1x TBS. Repeat this step 4x. After each wash, place the tube on a magnetic rack for 1 min, then discard the wash buffer.
      NOTE: Pierce Streptavidin Magnetic Beads are recommended as they minimize PEG-like background signals in mass spectrometry results.
    3. Pipette 1 mL of digested peptides into the bead-containing tube, mix gently, and then combine the mixture with the remaining sample in a 5 mL low-binding tube. Place the tube onto a multi-rotator and incubate for 1 h at RT.
    4. Transfer 1 mL of the mixture to a 1.5 mL tube and place it on the magnetic rack for 1 min. Discard the supernatant. Repeat this step until all beads are collected.
    5. Prepare 2 M urea in 50 mM ABC by dissolving 0.7207 g urea in 6 mL of 50 mM ABC. Wash beads with 1 mL of 2 M urea in 50 mM 2x and with 1 mL of TDW once. After each wash, place the tube on a magnetic rack for 1 min, then discard the wash buffer.
    6. Prepare 10% formic acid stock solution by dissolving 1 mL of formic acid in 9 mL of TDW. Prepare the elution buffer consisting of 80% acetonitrile and 0.1% formic acid in TDW. For example, mix 1.2 mL of acetonitrile, 15 µL of 10% formic acid stock, and 285 µL of TDW to make 1.5 mL elution buffer.
      CAUTION: Acetonitrile and formic acid are harmful due to their volatile properties, so wear a mask and be cautious when handling them.
    7. Pipette 100 µL of elution buffer into the tube, homogenize gently and then elute peptides using a thermoblock at 300 rpm for 5 min at 60 °C. After elution, place the tube onto a magnetic rack and transfer the eluate into a new 1.5 mL tube. Avoid taking beads when transferring the eluate. Repeat this step 5x.
    8. Place the tube containing 500 µL of eluate onto a magnetic rack for 5 min, then transfer it to a new 1.5 mL tube to ensure complete removal of the beads. The eluted samples can then be dried and used for mass spectrometry analysis.
    9. For additional information on LC-MS/MS analysis of enriched peptide samples and MS data processing, check Nguyen et al.20.

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Results

Example outputs are depicted in Figure 1, Figure 2, Figure 3, Figure 4 were previously published by Nguyen et al.20. Figure 1 illustrates the experimental procedure of TurboID-based PL in Drosophila ovary. First, a construct combining Zuc and TurboID was generated (Zuc-V5-TurboID). Well-known and distinct sub...

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Discussion

Here, we provide a comprehensive procedure for TurboID-based proximity labeling (PL) in the Drosophila ovary, developed through extensive troubleshooting to overcome various challenges. One key issue involved genetic constructs, as different fusion layouts significantly affected protein expression and biotinylation efficiency. For example, NES-V5-TurboID displayed weaker expression and biotinylation compared to other transgenes, occasionally complicating the interpretation of downstream assays. However, a revise...

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Disclosures

The authors declare no conflict of interest.

Acknowledgements

We thank the laboratory members for their discussions and help. This work was supported by the National Research Foundation (NRF) funded by the Korean government (MSIT) (RS-2024-00345327 to M.L.; RS-2023-00219563 to M.L.).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Antibodies
Goat anti-mouse IgG (H+L) secondary antibody, HRPInvitrogen31430Analytical grade, light-sensitive
Goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, alexa fluor plus 488InvitrogenA32731Analytical grade, light-sensitive
Goat anti-rabbit IgG (H+L) secondary antibody, HRPInvitrogen31460Analytical grade, light-sensitive
HSP90 antibody (rabbit)Cell Signaling Technology4874Polyclonal antibody
Pierce Streptavidin protein, HRPThermo Fisher Scientific21126Analytical grade
Streptavidin, Alexa Fluor 594 conjugateInvitrogenS11227Light-sensitive, analytical grade
V5-tag antibody (mouse)InvitrogenR960-25Monoclonal antibody
V5-tag antibody (rabbit) (D3H8Q)Cell Signaling Technology13202Monoclonal antibody
Fly LinesCompanyCatalog NumberComments/Description
pUASp Tom20 V5-TurboID/TM3Korea Drosophila Stock Center2373-2Fly stock
pUASp Zucchini V5-TurboID/CyoKorea Drosophila Stock Center2188Fly stock
pUASP_attB_V5-TurboID-NES/CyoKorea Drosophila Stock Center2635-4Fly stock
w[*]; P{w[+mC]=matalpha4-GAL-VP16}V2HBloomington Drosophila Stock Center7062Fly stock
Equipments and ConsummablesCompanyCatalog NumberComments/Description
Analytical BalanceMettler ToledoME2040.1 mg readibility
Amersham Protran 0.45 NC nitrocellulose western blotting membranes (300 mm × 4 m)Cytiva10600002-
Axygen 1.7 mL MaxyClear Snaplock Microcentrifuge TubeCorningMCT-175-CCo-polymer
BRAND Cover Glass (22 mm x 22 mm)Sigma-AldrichBR470055Borosilicate glass
Bulk Pipette Tips (10 µL)Vertex4110N00Propylene
Bulk Pipette Tips (1000 µL)Vertex4330N00Propylene
Bulk Pipette Tips (200 µL)Vertex4230N00Propylene
Cell Culture Plate (96-well)SPL300096Polystyrene, flat-bottom type
Centrifuge (with 1.5-2 mL tube-sized rotor)Eppendorf5424 RFixed-angle, refrigerated
Centrifuge (with 5 mL tube-sized rotor)Eppendorf5425 RFixed-angle, refrigerated
Chemical Spoon--Stainless steel
ChemiDoc Imaging systemBiorad12003153Western blot imaging system
CO2 fly pad--Anaesthetic board
CO2 tank & regulator--Caution of high pressure gas & dizziness
Compact incubatorJSRJSGI-10TCaution of high temperature
Confocal laser scanning microscopeZeissLSM 710Caution of intense laser beam
Conical tube (15 mL)SPL50015Polypropylene /High density polyethylene 
Conical tube (50 mL)SPL50050Polypropylene /High density polyethylene 
Digital balance---
Digital timer---
Dissection dish--Silicon
Dumont #5SF Forceps Fine Science Tools11252-00Super fine tips, caution of sharp object
DynaMag-2 magnetInvitrogen12321DFor 1.5-2 mL sized microcentrifuge tubes
Fisherbrand Superfrost Plus microscope slides (25 mm x 75 mm)Fisher Scientific12-550-15Caution of sharp object
Fly food bottles (with paper plug)Hansol TechFFB-21010-
Fly food vials (with cotton cap)Hansol TechFFV-11010-
Focused-ultrasonicatorCovarisM220Caution of ultrasonic wave
Gel knifeInvitrogenEI9010Stainless steel with plastic handle
Grade 3MM Chr blotting paper (46 cm × 57 cm)Cytiva3030-917Whatmann paper
Hand-held homogenizer and plastic pestleKimble749540-0000Cordless motor
Heat blockFine PCRALB6400Caution of high temperature
Lint-free tissueYuhan-Kimberly41112-
Liquid nitrogen--Caution of frostbite and burn
Microplate spectrophotometerAgilent TechnologiesEpochMonochromator-based optics
Microwave--Caution of high temperature
milliTUBE 1 mL AFA fiberCovarisPN 520135Glass
Mini gel tankThermo Fisher ScientificA25977Caution of high temperature
NuPAGE Bis-tris mini protein gels, 4–12%, 1.0–1.5 mm, 10-wellInvitrogenNP0323BOXContain polyacrylamide, caution of toxicity
NuPAGE Bis-tris mini protein gels, 4–12%, 1.0–1.5 mm, 15-wellInvitrogenNP0321BOXContain polyacrylamide, caution of toxicity
Paint brush (Ø 1.8 mm)Hwa hong791240-
Pipetman L (P1000L)GilsonFA10006MSingle channel micropipette with metal ejector
Pipetman L (P200L)GilsonFA10005MSingle channel micropipette with metal ejector
Pipetman L (P20L)GilsonFA10003MSingle channel micropipette with metal ejector
Pipetman L (P2L)GilsonFA10001MSingle channel micropipette with metal ejector
Power supplyMajor scienceMP-310Caution of high voltage electricity
Programmable rotatorBiosanRS-24-
Protein LoBind tubesEppendorf30108302-
Razor bladeDorco304020Stainless steel, caution of sharp object
Roller---
Serological pipette (10 mL)SPL91010Polystyrene
Serological pipette (25 mL)SPL91025Polystyrene
Serological pipette (5 mL)SPL91005Polystyrene
Serological pipette (50 mL)SPL91050Polystyrene
Snap cap low retention microcentrifuge tubes (1.5 mL)Thermo Scientific3451PKPolypropylene
Sponge padInvitrogenEI9052-
Stereo microscopeZeissStemi 508-
Stereo microscope Leica
ThermoMixer CEppendorf5382000015With 1.5 & 5 mL-sized blocks
TweezerAven Tools18526Plastic, flat tips
Vortex mixerScientific IndustriesSI-0236-
XCell II Blot moduleInvitrogenEI9051Caution of high temperature
XCell SureLock mini-cellInvitrogenEI0001Caution of high temperature
ChemicalsCompanyCatalog NumberComments/Description
AcetoneSigma-Aldrich650501Analytical grade
AcetonitrileSupelco1.00029.1000LC-MS grade, caution of inhalation toxicity and flammable
Albumin standard ampules (containing bovine serum albumin (BSA) at 2 mg/mL in 0.9% saline and 0.05% sodium azide)Thermo Scientific23209Part of Pierce BCA Protein Assay Kits (Thermo Scientific, 23225)
Albumin, bovine serumBioshopALB001Analytical grade
Ammonium bicarbonateSigma-Aldrich09830Analytical grade
Biotin Sigma-AldrichB4639Cell & tissue culture grade
Calcium chloride (CaCl2)Sigma-AldrichC5670Cell-culture grade
Clarity western ECL substrateBiorad1705060Analytical grade
DAPISigma-AldrichMBD0015Light-sensitive, analytical grade
Dithiothreitol (DTT)Thermo Fisher ScientificR0861Analytical grade
Fetal bovine serumCorning35-015-CVCell culture grade
Formic acidSigma-AldrichF0507Reagent grade, caution of inhalation toxicity
Grace's insect mediumGibco11595030Cell culture grade
Instant dry yeastNo BrandN1019-
IodoacetamideSigma-AldrichI1149Light-sensitive, analytical grade
MethanolCarlo Erba41484Analytical grade
Nail polish--Clear-type
Novex Tris-glycine SDS sample buffer (2X)InvitrogenLC2676Analytical grade
NuPAGE MOPS SDS Running Buffer (20X)InvitrogenNP0001Analytical grade
PageRuler Prestained protein ladderThermo Fisher Scientific26616Analytical grade
Phenylmethanesulfonyl fluoride Thermo Fisher Scientific36978Analytical grade
Phosphate buffer salineSigma-AldrichP4417Analytical grade
Pierce 16% Formaldehyde (w/v)Thermo Fisher Scientific28908Analytical grade, light-sensitive
Pierce BCA protein assay kitsThermo Scientific23225Analytical grade
Pierce BCA reagent A (containing sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1 M sodium hydroxide)Thermo Scientific23228Part of Pierce BCA Protein Assay Kits (Thermo Scientific, 23225)
Pierce BCA reagent B (containing 4% cupric sulfate)Thermo Scientific1859078Part of Pierce BCA Protein Assay Kits (Thermo Scientific, 23225)
Propionic acidSigma-Aldrich402907ACS grade
Protease inhibitor cocktail set IIICalbiochem539134Cell & tissue culture grade
SlowFade Gold Antifade MountantInvitrogenS36936Light-sensitive
Sodium chloride (NaCl)BioneerC-9025Analytical grade
Sodium deoxycholateSigma-AldrichD6750Analytical grade
Sodium dodecyl sulfateSigma-Aldrich71736Molecular grade
Sodium hydroxide (NaOH)BiosesangSR2018-100-00Analytical grade
Triple distilled water--Ultrapure (type 1)
Tris-buffered salineSigma-Aldrich94158Analytical grade
Tris-glycine transfer bufferKoma BiotechK0341001Analytical grade
Tris-HCl, pH 8BioneerC-9006Analytical grade
Triton x-100Sigma-Aldrich93443Molecular grade
TrypsinThermo Fisher Scientific20233Cell-culture grade
Tween 20Sigma-AldrichP9416Molecular grade
UreaSigma-AldrichU5378Molecular & cell-culture grade

References

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TurboID Proximity LabelingProtein Interaction MappingBiotinylated PeptidesMass SpectrometryGermline CellsProtein FoldingMembrane OrganizationVesicle TraffickingConfocal Imaging
In Vivo Application of TurboID-based Proximity Labeling in Drosophila melanogaster
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