The overall goal of the following experiment is to quantitate phospho peptide changes that occur within spermatozoa during sperm cell maturation. This is achieved by removing the coddle epididymus from the mouse, performing retrograde back flushing of the sperm cells and collecting the cells into a micro capillary tube. Next sperm cells are allowed to swim out into a balanced salt solution, which enables the removal of contaminating proteins, and then they're washed lyed and precipitated in order to remove contaminating salts and fats.
The gular proteins are digested using trypsin then enriched using titanium dioxide in order to enrich for phospho peptides. The results show quantitative changes in the phosphorylation status of proteins based on liquid chromatography, mass spectrometry analysis. This method can help answer key questions in the reproductive biology field, such as what are the phosphoproteomic changes as sperm traverse the EPIs or during capacitation.
Though this method has provided an insight into sperm cell biology, it can also be applied to model organisms, disease studies such as cancer, neurological pathologies, and general signal transduction pathways. Visual demonstration of this method is critical as it's difficult to determine the anatomy of the areas in which the EPIs will be manipulated. Begin by making 200 milliliters of Biggers, Whitten and Whitten or BWW working stalk solution by adding the following to one liter of Milli Q water to make a cannula.
Using polyethylene tubing with internal and external diameters of 0.4 millimeters and 1.1 millimeter respectively. Hold it over low heat until the tubing begins to melt. Immediately pull the ends of the tubing outward to stretch it, thus decreasing the outer diameter.
Cut the tubing to produce a narrowing of one end, which will allow for easier cannulation of the S deens. Cut the opposite end to approximately 15 centimeters. Then insert a 30 gauge needle into the blunt end, and attach a fully retracted three milliliter syringe to it.
Make a suction mouthpiece by cutting a 20 centimeter length of PE tubing and insert it into one end of the cannula. Insert the micro capillary glass tube holder and glass micro capillary. After euthanizing a mouse according to the text protocol, make a small incision in the scrotum to expose the epi.
Then use a pair of watchmakers. Number five forceps to pull the testis and epididymus out of the cavity. Cut to remove all but about one to two centimeters of the vast deference from the kata epididymus.
Then cut to remove the proximal EENT ducts and tissue connecting the epididymus to the testis and remove the entire male reproductive track under a dissecting microscope using between five x and 40 x magnification. Place one to two centimeters of the narrowed end of the cannula into the field of view and use tape to secure it with number five, watchmaker's forceps. Gently clasp each side of the vast deference and pull it over the visible portion of the cannula.
Then with non-absorbable black braided treated silk, tie a secure knot around the cannulated tissue using watchmakers. Number five forceps. Grab the distal end of the kata epi ides and remove the tunica Eugenia.
In order to expose a single epidermal tubule, gently expose the tubule and tease it apart to create an opening for the sperm to be released. Next, gently depress the plunger of the syringe to expel air into the vast deference. The pressure will cause spermatozoa to exit the broken tubule.
Then apply suction to the mouthpiece to draw the spermatozoa into the glass capillary. Gently blow into the mouthpiece or attach a syringe and expel the spermatozoa from the glass capillary into one milliliter of pre-war B WW solution. And use the solution to wash the cells three times to remove any contaminating proteins.
After removing the final wash, freeze the sperm for later use or use 4%chaps, two molar thio urea, and 50 millimolar tris pH 7.4 to solubilize the proteins incubate for one hour with intermittent vortexing. Then centrifuge the solution at 10, 000 GS for 20 minutes and transfer the supernatant to a new tube to reduce the disulfide bonds and alkylate the proteins at DTT at a final concentration of 10 millimolar to the protein solution. Vortex and incubate at room temperature for 30 minutes.
Then add 50 millimolar of IO acetamide to the lysate vortex and incubate for 30 minutes at room temperature in the dark. To precipitate the proteins, combine one volume of lysate, one volume of methanol, and 0.5 volumes of chloroform. After vortexing the sample, spin it at 10, 000 Gs for two minutes, collect the top layer, leaving about two millimeters to avoid aspirating the interface.
After adding one volume of methanol, gently invert the tube and spin again. Discard the supernatant and air dry the pellet for three to four minutes to carry out trypsin, digestion and phospho peptide enrichment. Begin by using 25 millimolar ammonium bicarbonate containing one molar urea.
To reconstitute the trypsin, combine F 50 to one weight per weight ratio of protein to the trypsin and incubate on a thermo mixer at 37 degrees Celsius and 700 RPM overnight. After spinning to pellet the undigested material, transfer the supernatant to a new tube. Using DHB buffer prepared according to the text protocol, dilute the trys inized peptides tenfold and apply it to 200 micrograms of dry titanium dioxide beads incubate on a rotator for one hour at room temperature after the incubation, use DHB buffer to wash the sample once more before using wash buffer consisting of 80%A CN and 2%TFA to wash the sample three times following the final centrifugation elute the phospho peptides by adding 2.5%ammonium hydroxide immediately after spinning and collecting the supernatant.
Use 0.3 microliters of formic acid to acidify the sample as seen here. One advantage of this protocol is that spermatozoa are isolated. In a quiescent state, many cells clump just after expulsion into B WW medium.
However, after 10 minutes, they become modal and the solution becomes homogeneous. The preparation described in this video typically produces 200 times 10 to the six zoa. This figure demonstrates the purity of spermatozoa from AR rat Cota epididymus.
One major issue concerning sample preparation is the yield from trips and digestion. Unlike TCA precipitation, which often requires the pH of the sample to be adjusted, phenol chloroform precipitation is quick and does not acidify the sample shown here is the protein pellet typically seen from 100 micrograms of sample between the lower and upper interface demonstrated. Here is reproducibility of this protocol.
The blue streaks appearing over time are peptides alluding from a C 18 nano column. As the concentration of acetonitrile increases in the modal population, there is a peptide cluster at about 651.5 Daltons that is completely absent in the non range. Once the phosphate peptide enrichment protocol is an efficient technique.
While attempting this procedure, it's important to remember that sample preparation is a key aspect to obtain reproducible results for proteomics. This method can be adapted to isolate glycoproteins, which can be used to answer additional questions such as which proteins undergo a change in the sic acid content of their proteome.
