The overall goal of this procedure is to efficiently extract proteins from the human cardiac mitral valve, suitable for proteomic analysis. This method can potentially help uncover pathogenic mechanisms in the field of cardiac valve disease, allowing for identification of novel diagnostic and prognostic disease markers and hopefully, of therapeutic targets. The main advantage of this protocol is that it provides an efficient extraction workflow compatible with many analytical applications.
The results being a more exhaustive characterization of the cardiac mitral valve proteum. Moreover, this protocol can also be applied to other systems, such as the porcine mitral valve, which bears a close resemblance to the human valve and is used in experimental model for valve function evaluations. Demonstrating the experimental procedure will be Stefania Ghilardi, a technician from my laboratory.
to prepare the human mitral valve, begin with an explanted heart that has been under cold ischemia for four to 12 hours. In a Cleanroom, remove the heart from the transport bag, and put it into a sterilized bucket. Next, transfer the heart to a biosafety cabinet.
There, using a disposable scalpel, cut perpendicularly to the major axis on the level of the left and right ventricles, about four centimeters from the apex. Then place the heart onto a sterile drape. Then, move aside the ascending aorta and pulmonary artery to access the left atrial roof.
On the left atrial roof, use forceps and picks to cut around the left auricle to expose the mitral valve. The mitral valve is fully contained in the left ventricle. Thus, expose the great mitral leaflet and the small mitral leaflet.
The anterolateral and the posteromedial commissures define the boarder of the anterior and posterior area. Now, with scissors and non-traumatic forceps, dissect the left atrium and the ventricle wall thickness that surrounds the mitral valve. During this dissection, identify the mitral aortic valve continuity.
Next, separate the anterior mitral valve leaflet from the posterior mitral valve leaflet by cutting along the commissures that boarder the posterior leaflet. When the procedure is completed, sanitize the table of the cabinet with a 70%isopropyl alcohol solution and a 6%hydrogen peroxide solution. Now, wash the posterior leaflet of the mitral valve in saline.
Then, cut the valve into small pieces, each less than a square centimeter. Wrap the pieces individually in aluminum foil, and snap freeze them with liquid nitrogen. To begin the protein extraction, use forceps to remove a sample from the liquid nitrogen, and immediately place it on dry ice.
Do not let the sample thaw. Next, into a dewar flask filled with liquid nitrogen, place the mortar, associated pestles, and the sample. It's critical that the liquid nitrogen is used to freeze the sample and to chill the grinder system.
This step prevents biological degradation and allows for efficient powdering, but requires technical training for safe handling. Once flash frozen, put the mortar and pestles into a polystyrene box containing dry ice. Also, place a spatula on the dry ice.
Next, remove the sample from the foil and load it into the mortar. And position the larger pestle above it. Using a screwdriver to rotate the pestle, grind the sample 15 to 20 times.
While grinding, mix the sample with the tip of a chilled spatula. After using the large pestle, repeat the grinding process with a smaller pestle. Obtaining a fine powder is critical to efficient protein extraction.
Now, dump the powdered sample into a 15 millileter centrifuge tube of known mass. Then brush any remaining material into the mortar using a cold spatula. Keep the tube with the sample on dry ice and quickly determine the mass of the sample.
Next, dump the sample into a glass homogenizer tube. Then, add 200 microliters of urea buffer for every 10 milligrams of sample. In doing this, recover the residuals left in the tube by rinsing them out with the urea buffer aliquot.
Now homogenize the sample using a motorized PFTE pestle running at 1, 500 rpm. Slowly press the pestle onto the sample with a twisting motion 10 times. Next, transfer the viscous yellow supernatant to a clean 1.7 milliliter centrifuge tube using a glass pipette.
Now repeat the homogenization on the remaining sample using half as much urea. Recover the supernatant and combine it with the previous collection. Next, place the tube on a tube rotator for 30 minutes.
After 30 minutes, load the tube into a cold centrifuge and spin down the sample at 13, 000 G for half an hour. Then, recover the supernatant. And measure the protein concentration using the Bradford protein assay.
After performing the prescribed protocol, the protein extract was studied using a variety of methods after some additional treatments. A total of 422 proteins were identified in the mitral valve tissue by two dimensional electrophoresis, liquid-phase isoelectric focusing, liquid chromatography-mass spectrometry, and 2D liquid chromatography-mass spectrometry. The 422 proteins were classified using Gene Ontology analysis.
The extracellular regions contained several expected proteins and other intracellular and cell surface proteins. Many of which were identified for the first time in mitral valves. The presence of four such proteins not previously identified in mitral valves was confirmed with antibodies in three unique samples.
The proteins are Septin-11, Four and a half LIM domains protein one, Dermatoponin, and alpha crystalline B.After watching this video, you should have a good understanding of how to extract proteins from the cardiac mitral valve for their identification and quantification. Once mastered, this protocol can be done in almost two hours if properly performed. While attempting this procedure, to get the largest yield of proteins with high protein integrity, it's vital that the samples does not thaw during any of the transfer.
