Identification of Mosquito Eggshell Proteins from Aedes aegypti by Liquid Chromatography with Tandem Mass Spectrometry (LC–MS/MS) Proteomic Analysis

1. Set up glass blood feeders wrapped in stretched Parafilm, connected to a water bath (40°C), and place the glass feeders on the screened top of the mosquito cage, secured with a lab clamp. Prewarm vertebrate blood (e.g., cow) to 37°C.

2. Load 1 mL of prewarmed vertebrate blood supplemented with 5 mm adenosine triphosphate into the feeders according to the manufacturer's instructions.

3. Allow 3- to 4-d-old adult female mosquitoes in a cage to feed on the blood source for 1 h.

4. Cold-anesthetize mosquitoes by placing the cage for 5 min in a refrigerator (4°C).

5. Place 100 cold-anesthetized mosquitoes onto a glass Petri dish on ice under a light microscope to facilitate sorting of engorged mosquitoes based on abdominal distension (Fig. 1). Use forceps to transfer fully engorged mosquitoes to a new cage. Follow standard rearing protocols (see Protocol: Aedes aegypti Culturing and Egg Collection [Clemons et al. 2010]; Protocol: Batch Rearing Aedes aegypti [Wohl and McMeniman 2023]; Imam et al. 2014). Maintain blood-fed mosquitoes with 10% sucrose (via soaked cotton balls) for several days.

• Although in general eggshell formation for each oocyte is completed ∼48 h after blood feeding, we recommend harvesting fully mature ovarian follicles at 4 d post–blood meal, which can be accomplished by depriving the gravid females of an oviposition substrate. Place the 10% sucrose-soaked cotton on the cage for only 1 h per day during this period to avoid mosquitoes ovipositing on the cotton.

6. On the day of dissection, cold-anesthetize mosquitoes as described in Step 4.

7. Place a drop of ice-cold PBS onto a glass slide. Place an anesthetized mosquito onto the drop of PBS. Under a light microscope, gently dissect ovaries from the mosquitoes by grasping the last two abdominal segments with forceps and pulling until the ovaries emerge (see Fig. 2 in Protocol: Visualization of Apoptotic Ovarian Follicles during Aedes aegypti Mosquito Egg Maturation by Fluorescent Imaging Studies (Isoe et al. 2023a).

• If this technique is not successful because of the size of the fully developed ovaries, the dorsal side of the abdomen can be cut along the longitudinal axis with microscissors and the ovaries gently lifted out.

8. Using a glass Pasteur pipette, transfer ovaries into a glass scintillation vial containing 5 mL of ice-cold PBS (Fig. 2).

9. Once approximately 100 pairs of ovaries are collected in the vial, pipette up and down 20 times. As soon as the mature elongated oocytes settle to the bottom of the vial, use a glass Pasteur pipette to remove all floating tissues and cells including inner, lateral, and common oviducts; trachea; muscles; germarium; secondary follicles; and follicular epithelial cells. Add 5 mL of ice-cold PBS and repeat Step 9 10 times to ensure a minimum contamination of nontarget cell types.

• Oocytes quickly descend because of gravity, whereas other ovarian tissues float or descend slowly.

10. Transfer the purified oocytes in 1 mL of ice-cold PBS into a Dounce homogenizer using a glass Pasteur pipette. Homogenize the oocytes on ice using 40 strokes in a Dounce homogenizer with pestle B. Let the insoluble eggshells form a pellet by gravity at the bottom of the glass homogenization tube. Remove the cloudy portion that contains vitellogenin yolk proteins and other soluble proteins with a glass Pasteur pipette. Add 1 mL of ice-cold PBS to resuspend the eggshell without homogenization, remove the cloudy portion, and repeat this process until the cloudiness disappears.

11. Transfer the homogenates containing eggshells onto a nylon mesh (40-µm) using a glass Pasteur pipette and wash thoroughly with 5 mL of PBS to remove oocyte cytosolic components. Repeat this washing process two additional times by pouring 5 mL of PBS onto the mesh to ensure a minimum contamination of cytosolic and membrane proteins (Fig. 2).

12. Add 1 mL of ice-cold PBS to the enriched eggshells on the mesh and quickly remove the PBS and eggshells with a glass Pasteur pipette. Transfer the enriched eggshells in PBS into a Dounce homogenizer using a glass Pasteur pipette. Remove the PBS and homogenize the eggshells in 5 mL of 6 m guanidine hydrochloride, pH 9 in a Dounce homogenizer with pestle A for 30 strokes. Place the homogenates in an incubator for 16 h at 37°C.

13. Precipitate the proteins by adding 45 mL of 100% ethanol and incubate the samples for 16 h at −20°C.

14. Pellet the eggshell proteins by centrifugation at 16,000g for 15 min at 4°C. Remove the supernatant carefully and wash the pellet with 90% ice-cold ethanol. Repeat the 90% ethanol wash two additional times to remove residual guanidine hydrochloride. Pellet the eggshell proteins by centrifugation at 16,000g for 15 min at 4°C after each wash.

15. Air-dry the pellets for 10 min to completely eliminate the ethanol.

16. Resuspend the eggshell proteins in 50 µL eggshell lysis buffer and use 2 µL of the resuspended protein to assay the protein concentration using the Pierce 660-nm reagent containing the Ionic Detergent Compatibility Reagent according to the manufacturer's instructions.

17. Add an equal volume of protein sample buffer to up to 20 µg of protein sample from Step 16 for a maximum of 50 µL and then denature the purified eggshell proteins in boiling H₂O for 5 min.

18. Load the mosquito eggshell protein samples onto an sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gel in Tris-Glycine-SDS protein electrophoresis buffer. Run the protein gels via a mini gel electrophoresis unit using standard techniques for ∼5 min until the proteins have entered the resolving gel (∼1 cm). This step is used to remove any nonprotein impurities.

19. Transfer the gel to a suitably sized gel staining box or large Petri dish and fix the gels with a 25-mL solution of 10% (v/v) glacial acetic acid and 50% (v/v) methanol for 30 min on a shaker table at room temperature. Stain the gel with 20 mL GelCode Blue Stain Reagent with shaking for 16 h at room temperature. Destain the gel using 25 mL ultrapure H₂O three times for ∼2 h at room temperature on a benchtop orbital shaker. This step enhances stain sensitivity.

20. Excise gel bands (∼1.0 cm²) on a clean glass plate using a clean surgical scalpel and transfer the gel bands into 1.7-mL microcentrifuge tubes. Destain bands in 1 mL of 50% (v/v) methanol in 50 mm ammonium bicarbonate at room temperature on a vortexer with a tube adapter at low speed for 1 h or until the stain is removed; change the solution several times if necessary. Remove the destain.

21. To reduce disulfide bonds in the protein sample, add 100 µL of 25 mm DTT in 50 mm ammonium bicarbonate to the 1.7-mL microcentrifuge tube containing the gel bands for 45 min at 60°C. Quickly vortex the tube. Remove the DTT and immediately add 100 µL of 55 mm iodoacetamide in 50 mm ammonium bicarbonate for 30 min at room temperature to alkylate-free cysteines.

22. Remove the solution and wash the bands in 1 mL of 50 mm ammonium bicarbonate on a vortexer with a tube adapter or shaker for 1 h at room temperature. Repeat this wash two times.

23. Cut the bands into small pieces with a razor blade and transfer the bands into a 1.7-mL microcentrifuge tubes. Dehydrate the bands by adding 500 µL of 100% acetonitrile and incubating for 10 min at room temperature. Remove as much acetonitrile as possible. Dry the samples in a SpeedVac concentrator to remove the remaining acetonitrile.

24. Add 30 µL of eggshell protease cocktail to the samples. Incubate for 16 h at 37°C.

25. Centrifuge the gel pieces for 2 min at 4000g at room temperature, and transfer the supernatant to a fresh 1.7-mL microcentrifuge tube. Add 50 µL (or just enough to cover the gel pieces) of solvent mixture for peptide extraction to the remaining gel pieces. Sonicate for 10 min at room temperature. Centrifuge for 2 min. Transfer the supernatant to combine with the original supernatant.

26. Repeat the extraction in Step 25, transfer the supernatant to a new 1.7-mL microcentrifuge tube, pool the supernatants, and completely dry the supernatants in a SpeedVac concentrator for 30 min at room temperature.

27. Identify eggshell proteins by detecting tryptic/lysC peptides using a mass spectrometer equipped with a nano-flow liquid chromatography system followed by matching them to databases containing theoretical protein digestions.

• In our laboratory, we use Thermo Scientific instruments, columns, and software. We have a Q Exactive Plus mass spectrometer incorporated with an EASY-Spray nanoESI source.

• i. Resuspend peptides in 10 µL of 0.1% formic acid (1.0 µg/µL).

• ii. Inject peptides (500 ng in 0.1% formic acid) onto an Acclaim PepMap 100 trap C18 Reversed Phase HPLC column, and elute peptides onto an Acclaim PepMap RSLC analytical column (separation column), using an H₂O (0.1% formic acid) and acetonitrile (0.1% formic acid) gradient over 120 min.

• iii. For MS, set the flow rate at 300 nL/min using a Dionex Ultimate 3000 RSLCnano System. Analyze peptides using a data-dependent method.

• iv. For MS/MS, use a normalized higher collision energy set at 27 and an isolation width of 1.5 m/z.

• v. Search MS and MS/MS raw data against the Ae. aegypti NCBI protein database and a common human-derived contaminant protein database using Proteome Discoverer software using a SEQUEST based search engine.

  • MS/MS spectra matches consider fully tryptic peptides with at most two missed tryptic cleavage sites. Variable post-translational peptide modifications are also considered (e.g., methionine oxidation and cysteine carbamidomethylation). To identify proteins at 95% confidence, use XCorr score cutoffs as determined by a reversed database search.

• vi. Further analyze the peptide and protein identification outputs with proteomics software (e.g., Scaffold Q + S), a program that uses Bayesian statistics to detect additional spectra.

• vii. Accept protein identifications that have at least two peptides at a 0.1% peptide false discovery rate with 95% protein confidence.

• viii. Subject the identified eggshell proteins to BLASTP protein searches against the nonredundant NCBI protein database to find possible functions.