This method can help answer key questions about dynamic protein synthesis and the proteome involved in the process. This technique allows identification of the mRNAs that are being selected for translation, the microRNAs that can participate in the process and interfere with translation, and the proteins that contribute to protein translation or are themselves being translated. This method can provide insight into cardiac responses to stress, but can also be applied to the study of acute responses to stress in other organs.
Demonstrating the procedure will be Miroslava Stastna, a project scientist from Jennifer Van Eyk's laboratory. On the day of the experiment, fill the left compartment of a gradient mixer with five milliliters of 15%sucrose gradient solution, and fill the right compartment with five milliliters of 50%sucrose gradient solution. Open the tap and switch on the pump to stir the two small compartments with a magnetic stirrer, and use a tube linked to the second tap to allow the mixed sucrose solutions to flow into a single ultracentrifuge tube with thin walls on ice.
Next, homogenize the fresh or frozen heart tissue with a POLYTRON in filtered lysis buffer for 15 seconds on ice. At the end of the incubation, centrifuge the lysate to remove the insoluble material, and harvest the supernatant. Set aside 50 microliters of the supernatant for protein determination, RNA isolation, and RNA integrity analysis, and layer 100 to 500 microliters of the remaining supernatant over the gradient solution.
Use fresh lysis buffer to balance the volumes between the two gradient tubes, and ultracentrifuge the samples in an ultracentrifuge with a swinging bucket rotor. Be very careful not to disturb the sucrose gradient before or after centrifugation. To collect the gradients, program the fraction collector to discard the first three minutes of collection and to collect the heavy, light, and non-translating fractions at the speed of one milliliter per minute in 17 fractions from four to 19 minutes with continuous monitoring of the absorbance at 254 nanometers.
Place each fraction on ice as soon as it is collected. Then store the samples at minus 80 degrees Celsius, if they are not being processed immediately. To extract proteins from the polysome fractions, combine the collected sucrose fractions into three final fractions, and mark the fractions as heavy, light, and non-translating.
Mix 0.5 milliliters of each sample with 60 microliters of 100%trichloroacetic acid, or TCA. Incubate the fractions overnight at minus 20 degrees Celsius in the dark. The next morning, thaw the samples on ice before pelleting the proteins by centrifugation.
Discard the supernatants, and wash the pellets two times with 0.5 milliliters of minus 20 degrees Celsius chilled acetone per centrifugation. Make sure pellet, after TCA precipitation, is completely dissolved, using pulse sonication to ensure complete solubilization, as necessary. Air dry the pellets after the last wash before resuspension in 360 microliters of Tris-HCl buffer.
Then add 40 microliters of dithiothreitol for a 45-minute incubation at 55 degrees Celsius with shaking. At the end of the incubation, add 50 microliters of iodoacetamide to the samples for a 30-minute incubation on the shaker at room temperature, protected from light, followed by digestion of the samples with a one-to-50 weight-to-weight trypsin-to-protein ratio for an overnight shaking incubation at 37 degrees Celsius. The next morning, after cooling, briefly centrifuge the samples, and adjust the pH to two to three with 10%formic acid to quench the trypsin activity.
To desalt the samples, first condition the sorbent in the wells of a 96-well plate with 200 microliters of methanol three times, followed by conditioning with 200 microliters of 0.1%formic acid per well three times. Next, load the samples onto each well, and wash the samples three times with 200 microliters of 0.1%formic acid per wash. Using gravitation, followed by low vacuum, elute the samples with 100 microliters of 0.1%formic acid in 50%acetonitrile two times into one low protein retention 0.5-milliliter tube per sample.
Then use a concentrator to evaporate the sample eluates to dryness, and reconstitute each tryptic peptide pellet in 50 to 100 microliters of 0.1%formic acid for mass spectrometry analysis. mRNA analysis can be performed to assess the distribution of a particular mRNA of interest in each fraction or for quantification and comparison of the combined polyribosomal translating fractions to the non-translating fraction as a ratio of mRNA abundance each fraction. For example, here, the changes in mRNA distribution across the translating and non-translating fractions after mouse heart subjection to ischemia or ischemia reperfusion, compared to sham heart treatment are shown.
In this representative experiment, the analysis of a subset of microRNAs in the pooled heavy fractions revealed that 22 microRNAs were associated with polysomes during ischemia, nine were associated during ischemia and reperfusion, and seven were common to both conditions, demonstrating that the association of microRNAs is dynamic in response to the stresses of ischemia and reperfusion. Further, the majority of mitochondrial ribosomal proteins were identified in the light fraction, and the majority of the cytosolic ribosomal proteins were identified commonly in both the heavy and light fractions. While attempting this procedure, it's important to remember to be very careful with the sucrose gradient.
TCA precipitation for extraction of proteins from polysome fractions was selected in our workflow based on both the ability to process the fraction with high concentration of sucrose and the number of proteins identified by mass spectrometry. Generally, individuals new to this method may struggle because of the challenges associated with the reproducibility of the sucrose gradient. TCA precipitation is advantageous since only small volumes is needed to precipitate the proteins, the pellet is visibile at the tube bottom, and there is no accumulation of sucrose during precipitation.
