The overall goal of this procedure is to isolate chromatin bound proteins from the mouse heart. This is accomplished by first homogenizing the heart and then isolating cardiac nuclei virus sucrose, density gradient. Next, next, the nuclear plasm is separated from the chromatin fraction.
Then proteins are extracted from the chromatin pellet to enrich for proteins loosely bound to the DNA. Finally, proteins are extracted from a different chromatin pellet to enrich for proteins tightly bound to the DNA. Ultimately, results can be obtained that show the characteristics of the nuclear proteome under different physiological conditions through quantitative label-free mass spectrometric analysis.
This method can help answer key questions in the cardiac field, such as how chromatin remodeling contributes to cardiac hypertrophy and failure. After sacrificing an adult mouse and excising the heart, rinse it in ice cold PBS. Then place it in a glass down with two milliliters of lysis buffer containing a protease and phosphatase inhibitor mixture and homogenize it.
Pour the lysates through a 100 micron strainer and collect the filtrate in a 1.5 milliliter tube from this point forward, unless otherwise specified, keep the sample on ice. Centrifuge the sample at 4, 000 RPM for five minutes at four degrees Celsius. Collect the supinate containing the cytosol and store it at minus 80 degrees Celsius.
Reeses bend the pellet in 200 microliters of lysis buffer. Add one milliliter of sucrose buffer to a new tube, and gently layer the resuspended pellet on top of the sucrose pad using a transfer pipette centrifuge at 5, 000 RPM for 10 minutes at four degrees Celsius. Remove the thin film on top as well as the sucrose pad, which contains the membrane.
Rinse the remaining pellet containing the nuclei with 200 microliters of ice cold P-B-S-E-D-T-A tate, the nuclei containing pellet in 200 microliters of detergent extraction buffer, and vortex the sample two times for 10 seconds each. Place the sample on ice for 10 minutes and then centrifuge it at 13, 000 RPM for five minutes at four degrees Celsius. Remove the supinate containing nucleoplasm proteins and store it at minus 80 degrees Celsius.
Rinse the pellets with ice cold P-B-S-E-D-T-A and resuspend it in 300 microliters of Tris, S-D-S-E-D-T-A buffer. Sonicate the sample three to six times for 10 seconds each. To break up the DNA, keeping the sample on ice between syndications centrifuge, 13, 000 RPM for five minutes of four degrees Celsius.
Keep the supinate containing the isolated protein and store at minus 80 degrees Celsius to enrich for proteins such as histones, tightly bound to DNA. Carry out a separate fractionation to isolate chromosome, beginning with an isolated adult heart or cardiomyocytes from rat PARPs Triturate the chromatin pellet in 400 microliters of 0.4 N sulfuric acid and vortex. To remove clumps, rotate the sample of four degrees Celsius for 30 minutes or overnight after the incubation, remove the nuclear debris by spinning at 16, 000 G for 10 minutes of four degrees Celsius.
Transfer the supinate to a fresh tube and add 132 microliters of trichloroacetic acid dropwise to the sample. Invert the tube several times and incubate on ice for 30 minutes. After spinning, discard the supinate and use ice cold acetone to gently rinse the histone containing pellet and spin.
Again, repeat the acetone, wash and air dry the pellet before re suspending it. In 100 microliters of Triss S-D-S-E-D-T-A buffer. Use one molar triss to adjust the pH to eight.
Sonicate the sample in a water bath for 15 minutes. Store the sample at minus 80 degrees Celsius. Use mass spectrometry to carry out analyses to determine purity and to identify proteins shown here.
The overlaid extracted ion chromatograms for a single peptide that has been mapped to the HMGB one protein. Each chromatogram comes from a different chromatin sample and has been color coded to represent the physiological state of the mouse from which the sample was acquired. Three biological replicates were analyzed for each of the physiological conditions, basal cardiac hypertrophy and heart failure with each sample being analyzed twice, giving a total of 18 chromatograms grams.
By integrating the area under the curve, the relative abundance can be determined for each replicate. In this panel, the six replicates have been averaged for each of the three physiological conditions to give overall abundance values. It is clear from this analysis that the abundance of HGB one changes during the course of the disease as seen here.
The fragmentation spectrum for this peptide confirms its amino acid sequence and enables it to be mapped to the H MG B one protein. In contrast, quantitation of a different peptide from the protein histone H four shows that its abundance does not change with disease. We can analyze the reproducibility of the changes in peptide abundance that we observe between the different physiological states using statistical analysis.
First, the abundance of all peptides identified in each replicate is subjected to anova, followed by principal component analysis or PCA. The clustering of both technical and biological replicates in this plot confirms that the abundance changes detected by mass spectrometry are consistent and reproducible between the different disease states. Basal in blue hypertrophy red failure in green of equal importance as the quantification and reproducibility of the results is the increased coverage of the nuclear proteome enabled by nuclear sub fractionation.
This figure illustrates that analysis of the whole nuclear alone fails to uncover even a majority of the nuclear proteins identified when combining individual analysis from the separate compartments. Furthermore, the moderate overlap between the different fractions demonstrates the biological relevance of considering these compartments individually for added insight into nuclear function and gene regulation. Finally, the ability to specifically fractionate acid extracted or detergent extracted chromosome enriches for key chromatin structural proteins such as histones, thereby enabling more focused mass spectrometric analysis on a group of proteins without requiring specific antibodies for each variant and isoform While attempting this procedure, it's important to remember to keep any samples that will be analyzed on the mass spectrometer free of keratin contamination.
This is especially important in subsequent steps following the protein isolation.
