The overall goal of this procedure is to determine the gender specificity of differences in the proteome profiles of skeletal muscle. This is accomplished by first mincing and thoroughly homogenizing biceps brachy eye tissues from male and female mice in order to extract most proteins. Next, the sample is applied to an IEF gel strip, and proteins are separated based on their isoelectric point, followed by an SDS page, separation in the second dimension according to size, following staining and imaging of the gels, the proteomic profiles of male and female muscle samples are compared and spots with significant differences in abundance between the two genders are excised.
Finally, the proteins in the gel plugs are digested with trypsin. The resulting peptides are extracted from the gel and subjected to nano liquid chromatography, mass spectrometry analysis for identification. Ultimately, results can be obtained that show differential expression of proteins in two or more experimental conditions through two dimensional gel electrophoresis and mass spectrometry based proteomics.
Though this method can provide insight into muscle gender specificity, it can also be applied to other cells, tissues, organs, or fluids in varying physiological states. Demonstrating the procedure will be Dr.Kalina Diva, the technical director of the Smith College Center for proteomics. After isolating cleaning and mincing mouse skeletal muscle at 45 microliters of extraction, buffer pergram of tissue, refer to the written protocol for this and all other buffer recipes using a motorized pestle, homogenize the tissue seven times for 15 seconds each at four degrees Celsius with two minutes cooling on ice between each homogenization.
Centrifuge the samples at 15, 800 Gs for 30 minutes at four degrees Celsius. Save the supernat and measure its volume. Discard the pellet to precipitate the proteins at three volumes of ice.
Cold reagent grade acetone per volume of supernatant. Vortex the tubes for 15 seconds, then centrifuge at 15, 800 Gs for 10 seconds to form pellets. Discard the supernat and re suspend the pellets and extraction buffer with the motorized pestle and polypropylene stirring rods precipitate the proteins a second time by adding an additional three volumes of acetone.
After resus suspending the pellet and extraction buffer, determine the protein concentration and or store the samples at minus 20 degrees Celsius. To perform isoelectric focusing of the proteins, remove a ready strip IPG strip from the minus 20 degrees Celsius freezer and allow it to equilibrate at room temperature for 10 to 15 minutes. Vortex the sample and pipette 600 to 800 micrograms of protein in a straight line at the back edge of a channel in the sample rehydration tray, leaving about one centimeter at each end.
Using forceps, peel off the plastic cover of the IPG strip, making sure to handle only the ends of the strip and avoiding any contact with the gel. Note the positive marked end of the strip and position it at the left side of the tray. Place the IPG strip gel side down on top of the sample.
Avoid trapping bubbles underneath. Allow it to rehydrate for one hour. Overlay the strip with 2.5 milliliters of mineral oil.
Cover the tray with a lid and allow it to rehydrate overnight. At room temperature the next day, place a paper wick at both ends of the focusing trade channel and wet each one with 10 microliters of millipore water. Remove the IPG strip and hold it vertically for about 10 seconds.
To drain the oil, blot the plastic backing with a Kim wipe. Place the IPG strip gel side down with the basic end to the left. In the focusing tray, cover the strip with 2.5 milliliters of mineral oil.
Position the tray into the protean IEF cell and program a three step protocol at a default cell temperature of 20 degrees Celsius, a maximum current of 50 microamps per strip and no rehydration time. After the program is complete, transfer the strip gel site up to an 11 centimeter e equilibration tray. Add a four milliliters of reduction buffer to the channel and equilibrate the strip for 10 minutes with mild shaking.
Remove the reduction buffer and add four milliliters of alkylation buffer to the channel and equilibrate the strip with shaking for an additional 10 minutes. Rinse the IPG strip by briefly dipping it in one XSDS running buffer. Next, lay the strip gel side up onto the back plate of an 11 centimeter criterion.
Precast 10.5 to 14%tris H-C-L-S-D-S poly acrylamide gel and gently push it in so that it makes full contact with the SDS gel. Make sure no bubbles are trapped between the two gel surfaces. Overlay the IPG strip with one milliliter of molten agro solution and let it solidify for five minutes.
Then load five microliters of protein marker into the single. Well run the gel in SDS buffer at 200 volts at room temperature, or at four degrees Celsius. When the dye front reaches the bottom of the gel, remove it from the plastic cast and stain it overnight by gently shaking it in Kumasi Brilliant blue.
Shake the gel in detain one solution two times for three hours each and detain two overnight. Store the gel in 7%acetic acid for analysis. After imaging the gel carrying out a comparative analysis of the spot patterns between gels and cutting out the spots of interest at 200 microliters of detaining solution to the gel plugs vortex and incubate the samples at room temperature for 30 minutes with shaking.
Then carefully remove and discard the solution and repeat. Add 30 microliters of freshly prepared reducing buffer to the tubes containing the gel plugs and incubate at 60 degrees Celsius for 10 minutes. Allow the samples to cool.
Then remove and discard the buffer. Add 30 microliters of Alkylation buffer and incubate the samples in the dark at room temperature For one hour, remove and discard the alkylation buffer. Wash the gel plugs by adding 200 microliters of detaining solution to each tube and incubate the samples at 37 degrees Celsius for 15 minutes.
Remove and discard the detaining solution and repeat the wash. Next, add 50 microliters of aceto nitrile to the gel plugs and incubate them for 10 minutes at room temperature to allow them to shrink dry. Then carefully remove the aceto nitrile and repeat the gel pieces.
Should look white and small. Allow the gel pieces to dry in a concentrator for 10 minutes. Swell the gel pieces by adding 10 microliters of activated tripsin solution.
Incubate at room temperature for 15 minutes. Add 25 microliters of digestion buffer to the tubes. Incubate the samples at room temperature overnight with shaking sonicate the samples for 10 minutes and spin them down before transferring the supernatant to a fresh tube.
To further extract peptides, add 10 microliters of 0.1%formic acid to the plugs and incubate for five minutes at room temperature with shaking sonicate the samples for 10 minutes. Collect the supernatant and combine it with the previously saved supernatant. The proteins can then be desalted and prepared for HPLC coupled mass spectrometry male and female unexercised mirroring skeletal muscle was extracted and separated into a two dimensional map.
The first level of the muscle comparative proteome once visualized using high resolution digital imaging, about 800 protein spots were detected in each as seen here. Using the male proteome map as a baseline, it is clear that there are numerous spots that change in abundance in the female proteome spots that increased greater than or equal to two fold in females are circled red, and those that decreased greater than or equal to twofold are circled blue. The identities of the protein spots that change more than twofold up or down and are statistically significant with an N of five mice are determined by amino acid sequence analysis using liquid chromatography coupled mass spectrometry.
In this plot, the x axis represents female mice pre-exercise, and the Y axis represents a single bout zero hour time point group. The results show that females demonstrate an abundant decrease in sugar energy metabolism, enzymes, and in creatine kinase enzyme isoforms a different energy supply system in muscles. Both humans and animals display higher male serum CK levels at rest and following exercise, but serum CK levels do not necessarily correlate with the amount of myo fibrillar.
This gender dimorphism in the cellular abundance of CK in miren biceps brachii muscle is a novel finding and may answer the physiologically different serum CK levels. Following this procedure, other methods like quantitative immuno blotting can be performed in order to verify the results. After watching this video, we should have a good understanding of how to prepare and separate samples for differential proteome profiling.
