Name: optimized protocol for the extraction of proteins from the human mitral valve
Uploaded: 2017-06-14T14:00:00
Description: Watch this Scientific Journal Video about Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve at JoVE.com

The Italian Ministry of Health supported this study (RC 2013-BIO 15). We thank Barbara Micheli for her excellent technical assistance.

In this protocol, the human hearts are collected during multiorgan explantation (cold ischemia time of 4-12 h, mean 6 &#177; 2 h) from multi-organ donors excluded from organ transplantation for technical or functional reasons, despite normal echocardiographic parameters. They are sent to the Cardiovascular Tissue Bank of Milan, Monzino Cardiologic Center (Milan, Italy) for the banking of the aortic and pulmonary valves. The mitral posterior leaflets are not used for clinical purposes, so they are collected during the aor...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+and+highly+sensitive+proteome+coverage+by+LC-MS/MS+without+prefractionation.">Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation.</a> Mol Cell Proteomics. 10 (8), M110.003699 (2011).</li><li>Loardi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+mitral+valve+prolapse:+the+harvest+is+big,+but+the+workers+are+few.">Biology of mitral valve prolapse: the harvest is big, but the workers are few.</a> Int J Cardiol. 151 (2), 129-135 (2011).</li><li>Schoen, F. 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One critical step of this protocol is the use of liquid nitrogen to freeze the sample and to chill the grinder system. The use of liquid nitrogen prevents biological degradation and allows for efficient powdering, but it requires specific training for safe handling.
In this protocol features a grinder system for sample grinding because small samples are difficult to recover from standard mortar and pestles. In this case, small samples spread as a fine powder over the mortar surface, rendering ...

Recent evidence has altered the understanding of the roles of the many regulatory mechanisms that occur after mRNA synthesis. Indeed, translational, post-transcriptional, and proteolytic processes can regulate protein abundance and function. The dogma &#8211; which says that mRNA concentrations are proxies to those of the corresponding proteins, assuming that transcript levels are the main determinant of protein abundance &#8211; has been partially revised.Indeed, transcript levels only partially predict protein abundance, suggesting that post-transcriptional events occur to regulate the proteins within cells1,2.
Furthermore, proteins ultimately dictate the function of the cell and therefore dictate its phenotype, which can undergo dynamic changes in response to autocrine, paracrine, and endocrine factors; blood-borne mediators; temperature; drug treatment; and disease development. Thus, an expression analysis focused on the protein level is useful to characterize the proteome and to unravel the critical changes that occur to it as part of disease pathogenesis3.
Therefore, the opportunities that proteomics present to clarify health and disease conditions are formidable, despite the existing technological challenges. The particularly promising areas of research to which proteomics can contributeinclude: the identification of altered protein expression at any level (i.e., whole cells or tissue, subcellular compartments, and biological fluids); the identification, verification, and validation of novel biomarkers useful for the diagnosis and prognosis of disease; and, hopefully, the identification of new protein targets that can be used for therapeutic purposes, as well as for the assessment of drug efficacy and toxicity4.
Capturing the complexity of the proteome represents a technological challenge. The current proteomic tools offer the opportunity to perform large-scale, high-throughput analysis for the identification, quantification, and validation of altered protein levels. In addition, the introduction of fractionation and enrichment techniques, aimed at avoiding the interference caused by the most abundant proteins, has also improved protein identification by including the least abundant proteins. Finally, proteomics has been complemented by the analysis of post-translational modifications, which progressively emerge as important modulators of protein function.
However, the sample preparation and protein recovery in the biological specimens under analysis still remain the limiting steps in the proteomic workflow and increase the potential for possible pitfalls5. Indeed,in most of the molecular biology techniques that must be optimized, the first steps are tissue homogenization and cell lysis, especially during the analysis of low-abundance proteins for which amplification methods do not exist. In addition, the chemical nature of proteins can influence their own recovery. For example, the analysis of highly hydrophobic proteins is very challenging, because they easily precipitate during isoelectric focusing, while trans-membrane proteins are almost insoluble (reviewed in Reference 5). Furthermore, the tissue composition variability creates a significant barrier to developing a universal extraction method. Finally, because almost all of the clinical specimens are of limited quantity, it is essential to enable protein preparation with maximal recovery and reproducibility from minimal sample amounts6.
This work describes an optimized protocol for protein extraction from the normal human cardiac mitral valve, which represents a very challenging sample for proteomic analysis. The normal mitral valve is a complex structure lying between the left atrium and the left ventricle of the heart (Figure 1). It plays an important role in the control of blood flow from the atrium to the ventricle, preventing backflow and ensuring the proper level of oxygen supply to the whole body, thus maintaining an adequate cardiac output. However, it is often considered to be an &#34;inactive&#34; tissue, with a low cellularity and few components, mainly in the extracellular matrix. This is because, in normal conditions, the resident valvular interstitial cells (VICs) present a quiescent phenotype with a low protein biosynthesis rate7.
However, it has been demonstrated that, in a pathological state, the number of VICs in the spongiosa increases and their protein synthesis is activated, together with other functional and phenotypical changes8. Therefore, it is not surprising that the minimal data available in the literature focus on the analysis of pathological mitral valves9,10, in which the increased number of activated VICs might explain the relatively high number of identified proteins.
In conclusion, the present protocol may serve to develop the understanding of the pathogenic mechanisms responsible for mitral valve diseases through the study of mitral valve protein components. Indeed, a greater understanding of the underlying pathological processes could help to improve the clinical management of valve diseases, whose current indications for intervention are largely predicated on hemodynamic considerations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Saline solution</td>
			<td></td>
			<td></td>
			<td>0.9 % NaCl</td>
		</tr>
		<tr>
			<td>Eurocollins A</td>
			<td>SALF</td>
			<td>30874046</td>
			<td>Balanced organ&#39;s transport medium. Combine 400 mL of Eurocollins A with 100 mL Eurocollins B to obtain balanced medium Eurocollins</td>
		</tr>
		<tr>
			<td>Eurocollins B</td>
			<td>SALF</td>
			<td>30874022</td>
			<td>Balanced organ&#39;s transport medium. Combine 400 mL of Eurocollins A with 100 mL Eurocollins B to obtain balanced medium Eurocollins</td>
		</tr>
		<tr>
			<td>Wisconsin</td>
			<td>Bridge life</td>
			<td>RM/N 4081</td>
			<td>Balanced organ&#39;s transport medium</td>
		</tr>
		<tr>
			<td>Biohazard vertical flow air</td>
			<td>Burdinola</td>
			<td></td>
			<td>Class A GMP classification</td>
		</tr>
		<tr>
			<td>Dewar Flask</td>
			<td>Thermo Scientific</td>
			<td>Nalgene 4150-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogrinder system</td>
			<td>OPS diagnostics</td>
			<td>CG 08-01</td>
			<td>Grinder system containing mortars, pestles and screwdriver</td>
		</tr>
		<tr>
			<td>Stainless steel forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable sterile scalpel</td>
			<td>Medisafe</td>
			<td>MS-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel scissors</td>
			<td></td>
			<td></td>
			<td>Autoclavable</td>
		</tr>
		<tr>
			<td>Stainless steel picks</td>
			<td></td>
			<td></td>
			<td>Autoclavable</td>
		</tr>
		<tr>
			<td>Disposable sterile drap</td>
			<td>Mon&#38;Tex</td>
			<td>3.307.08</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizing solution with isopropyl alcohol</td>
			<td></td>
			<td></td>
			<td>70% isopropyl alcohol</td>
		</tr>
		<tr>
			<td>Sterilizing solution with hydrogen peroxide</td>
			<td></td>
			<td></td>
			<td>6% hydrogen peroxide</td>
		</tr>
		<tr>
			<td>Micropipette, 1 mL, with tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>VWR international</td>
			<td>9278</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL centrifuge tubes</td>
			<td>VWR international</td>
			<td>PIER90410</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea buffer</td>
			<td></td>
			<td></td>
			<td>8 M urea, 2 M thiourea, 4 % w/v CHAPS, 20 mM Trizma, 55 mM Dithiotreitol</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma aldrich</td>
			<td>U6504-1KG</td>
			<td>To be used for Urea buffer</td>
		</tr>
		<tr>
			<td>Thiourea</td>
			<td>Sigma aldrich</td>
			<td>T8656</td>
			<td>To be used for Urea buffer</td>
		</tr>
		<tr>
			<td>CHAPS</td>
			<td>Sigma aldrich</td>
			<td>C3023-5GR</td>
			<td>To be used for Urea buffer</td>
		</tr>
		<tr>
			<td>Dithiotreitol</td>
			<td>Sigma aldrich</td>
			<td>D0632-5G</td>
			<td>To be used for Urea buffer</td>
		</tr>
		<tr>
			<td>Syringe 50 mL</td>
			<td>PIC</td>
			<td></td>
			<td>To be used to filter Urea buffer</td>
		</tr>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td>To be used to filter Urea buffer</td>
		</tr>
		<tr>
			<td>PFTE Pestle, 2 mL</td>
			<td>Kartell</td>
			<td>6302</td>
			<td>Part of Potter-Elvehjem homogenizer</td>
		</tr>
		<tr>
			<td>Borosilicate glass mortar</td>
			<td>Kartell</td>
			<td>6102</td>
			<td>Part of Potter-Elvehjem homogenizer</td>
		</tr>
		<tr>
			<td>Stirrer</td>
			<td>VELP scientifica</td>
			<td>Stirrer DLH</td>
			<td>To be used for homogenization by Potter-Elvehjem</td>
		</tr>
		<tr>
			<td>Bradford Protein assay</td>
			<td>Bio-Rad laboratories</td>
			<td>5000006</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube rotator</td>
			<td>Pbi International</td>
			<td>F205</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene box</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>For centrifugation of 1.7 mL centrifuge tubes at 13,000 x g</td>
		</tr>
		<tr>
			<td>Freezer -80&#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Precision balance</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td></td>
			<td></td>
			<td>For sterilization</td>
		</tr>
		<tr>
			<td>Cryogenic gloves for liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Professional forced ventilation and natural air convection oven</td>
			<td></td>
			<td></td>
			<td>For sterilization</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma aldrich</td>
			<td>P8340-5ML</td>
			<td>100X solution</td>
		</tr>
		<tr>
			<td>ProteoExtract Protein Precipitation Kit</td>
			<td>Calbiochem</td>
			<td>539180</td>
			<td></td>
		</tr>
		<tr>
			<td>RapiGest</td>
			<td>Waters</td>
			<td>186001861</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytoscape</td>
			<td>www.cytoscape.org</td>
			<td>version 2.7</td>
			<td>Software platform for Gene Ontology analysis</td>
		</tr>
		<tr>
			<td>BiNGO</td>
			<td>http://apps.cytoscape.org/apps/bingo</td>
			<td>version 3.0.3</td>
			<td>Plugin for Gene ontology analysis</td>
		</tr>
		<tr>
			<td>AlphaB Crystallin/CRYAB Antibody</td>
			<td>Novus Biologicals</td>
			<td>NBP1-97494</td>
			<td>Mouse monoclonal antibody against CryAB</td>
		</tr>
		<tr>
			<td>Septin-11 Antibody</td>
			<td>Novus Biologicals</td>
			<td>NBP1-83824</td>
			<td>Rabbit polyclonal antibody against septin-11</td>
		</tr>
		<tr>
			<td>FHL1 Antibody</td>
			<td>Novus Biologicals</td>
			<td>NBP-188745</td>
			<td>Rabbit polyclonal antibody against FHL-1</td>
		</tr>
		<tr>
			<td>Dermatopontin Antibody</td>
			<td>Novus Biologicals</td>
			<td>NB110-68135</td>
			<td>Rabbit polyclonal antibody against dermatopontin</td>
		</tr>
		<tr>
			<td>Goat Anti mouse IgG HRP</td>
			<td>Sigma aldrich</td>
			<td>A4416-0.5ML</td>
			<td>Secondary antibody for immunoblotting</td>
		</tr>
		<tr>
			<td>Goat Anti rabbit IgG HRP</td>
			<td>Bio-Rad laboratories</td>
			<td>170-5046</td>
			<td>Secondary antibody for immunoblotting</td>
		</tr>
	</tbody>
</table>

optimized protocol for the extraction of proteins from the human mitral valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The extraction and dissolution of proteins in the urea buffer is directly compatible with proteomic methods based on isoelectrofocusing (two-dimensional electrophoresis (2-DE)11 and liquid-phase isoelectric focusing (IEF)12) and with immunoblotting after dilution in Laemmli buffer13 containing a protease inhibitor cocktail14.
For gel-...

Watch this Scientific Journal Video about Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve at JoVE.com

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

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.

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