意大利卫生部支持这项研究（RC 2013-BIO 15）。我们非常感谢Barbara Micheli的优秀技术援助。

在该方案中，尽管正常的超声心动图参数，在多器官移植（冷缺血时间4-12小时，平均6±2小时）内，从器官移植排除器官移植的技术或功能原因收集人心脏。他们被送到米兰的心血管组织银行Monzino心脏中心（意大利米兰），为主动脉瓣和肺动脉瓣的银行服务。二尖瓣后叶不用于临床目的，因此在从供体亲属获得知情同意后，在主动脉瓣和肺动脉瓣隔离期间收集二尖瓣后叶。移植和研究的组织仅在家长同意后收集;在同意书上，只有在不适合人类临床使用（ 即微生物，功能和血清学问题）的情况下，他们授权（或不）使用心脏组织进行研究，遵循伦理委员会的指导方针蒙齐诺心脏中心。 二尖瓣制备<ol><li>器官移植后尽早收集人体二尖瓣（冷缺血时间4-12 h）。 </li><li>在洁净室内，从含有冷（4℃）溶液（ 即盐水溶液或平衡介质Eurocollins或威斯康星州）的运输袋中取出心脏。将其放入桶中并放置在生物安全柜（生物危害垂直空气流，A级，良好生产规范（GMP）分类）中，以进行阀门准备。 </li><li>将心脏放在柜内的无菌一次性悬垂物上。使用无菌的一次性手术刀，在距离顶点约4厘米处的左心室和右心室的水平上将心脏完全切开，与其主轴垂直。 </li><li>移动升主动脉和肺动脉显示左心房顶。 </li><li>用无菌高压灭菌镊子和捡拾，切割左耳廓上的左心房顶部，使二尖瓣可见，并允许确定大二尖瓣（前）和小二尖瓣（后）。 注意:前外侧和后内侧合缝定义前叶和后区的边界。 </li><li>使用无菌高压灭菌剪刀和非创伤镊子，解剖左心房和围绕整个二尖瓣周围的心室壁厚度。 </li><li>确定主动脉瓣主动脉瓣连续性。 注意:左心室包含整个二尖瓣和和弦。 </li><li>将二尖瓣二尖瓣分离自二尖瓣后叶小叶，沿着插入与心室切开后叶（连合）。 </li><li> 在盐水溶液中洗后叶。将小叶切成小块（&lt;1厘米2 ），并单独包裹在铝箔上。用液氮快速冻结。 注意:使用液氮时应遵循组织安全程序。 <ol><li>在程序结束时，用70％异丙醇溶液和6％过氧化氢溶液对柜子进行消毒。 </li></ol></li></ol>蛋白质提取<ol><li>使用镊子拿起储存在液氮中的样品，并立即将其放在干冰上，同时仍然包裹在铝箔中。在转运期间不要让样品解冻。 </li><li>在研磨之前，将研磨机系统（ 例如， CryoGrinder）的瓷/锆砂浆和样品与样品一起放入含有液氮（〜500 mL）的杜瓦瓶中。 注意:使用液氮时应遵循组织安全程序。 </li><li>将砂浆和杵放在含干冰的聚苯乙烯箱中。从铝箔上取出样品并放入砂浆中。 </li><li> 研磨他用大杵取样15-15次，用螺丝刀旋转杵。在研磨过程中将样品与预先冷却的铲刀的尖端混合。 <ol><li>用小杵重复。 </li></ol></li><li> 将研磨的样品转移到先前称重的管（ 例如 15mL离心管）上，通过倒置管，将其放置在研钵上，并将它们反转到一起以将样品移动到管中。使用预冷的铲子从砂浆中回收所有材料。 <ol><li>将样品保持在干冰上，以避免样品在转移过程中解冻。 </li></ol></li><li>计算样品的净重。 </li><li>在每个样品后清洁砂浆和杵，并通过高压灭菌或在200℃加热2小时对其进行去污。 </li><li>通过反转将粉末样品从离心管转移到均化器的玻璃管中。 </li><li>加入过滤的尿素缓冲液将r（8M尿素，2M硫脲，4％w / v CHAPS，20mM Tris和55mM二硫苏糖醇）加入到玻璃管中，每10mg粉状组织200μL尿素缓冲液。 注意:离心管中残留的粉末样品可以使用部分计算的尿素缓冲液体积进行回收。 </li><li>使用装有硼硅酸盐玻璃研钵和聚四氟乙烯（PTFE）杵的搅拌器均匀化样品。用搓动（1500转/分钟）将杵慢慢按压到样品上10次。 </li><li>回收上清液并将其转移到干净的1.7 mL离心管中。再次用新鲜的尿素缓冲液提取剩余的样品，加入第一次提取过程中使用的体积的一半。 </li><li>重复步骤2.10。 </li><li>回收上清液并将其与步骤2.11的上清液组合。将合并的上清液放在管旋转器上30分钟。 </li><li>在13,000 xg和4℃下离心管30分钟。 </li><li>恢复上清液d根据制造商的说明书使用Bradford蛋白测定法测量蛋白质浓度。将样品储存在-80°C直到使用。 </li></ol>

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作者没有什么可以披露的。

该方案的一个关键步骤是使用液氮冷冻样品并冷却研磨机系统。液氮的使用可防止生物降解并有效地进行粉末化，但需要对培训进行安全处理。 在该协议中，具有用于样品研磨的研磨机系统，因为小样品难以从标准的砂浆和杵中回收。在这种情况下，小样品作为细粉扩散到砂浆表面，使收集困难。另一个优点是研磨机是电动的，这允许以可再现的方式处理更多数量的样品并且没有增加的疲劳。对研磨机的使用的一个限制是，必须将杵的力量小（100mg或更少）的样品的尺寸有效地压在砂浆上。此外，研磨机组件必须在清洁用途之间加热至室温<html> G。因此，该过程是耗时的，并且如果每天处理许多样品，则需要许多组。 另外的关键步骤是提取缓冲液的制备。盐浓度，特别是尿素（8M）和硫脲（2M）的浓度相当高;因此，盐的体积几乎是溶液总体积的一半。此外，考虑到必须避免热量，溶解是不容易的，因为在大于37℃时，尿素可导致蛋白质/肽的N末端和赖氨酸和精氨酸残基17的侧链氨基处的蛋白质氨基甲酰化。一旦溶解，尿素缓冲液必须用0.22μm过滤器过滤，并且可以在-80℃下储存4周而不影响其提取效果，但是在使用之前必须将其温热至15℃以上才能完全溶解。 修改和故障排除在本协议中，使用所述的尿素缓冲液进行蛋白质提取，因为它是用于蛋白质组研究的最常用的蛋白质提取溶液之一，因为其与等电聚焦的相容性及其在溶解微溶蛋白中的效率18已经被证明这种缓冲液可以非常有效地溶解微溶蛋白，例如整合膜蛋白19或高度易于聚集的蛋白质，如微管蛋白18 ，此外，该缓冲液与Bradford测定法完全相容以确定蛋白质浓度，它可以直接用于2-DE和液相IEF分析。 然而，该缓冲液对于溶解样品中存在的所有蛋白质不是理想的。不同的提取缓冲液有可能揭示该方案不可检测的蛋白质。例如，它是很好的kno可以用酸提取或三氯乙酸/丙酮沉淀20更好地提取核糖体和核蛋白，而碱性pH水平更适合于膜蛋白21,22 。因此，使用替代缓冲液可能需要额外的蛋白质沉淀步骤来消除干扰2-DE或液相IEF的盐。 技术的局限性通过该方案鉴定的蛋白质数量相对较少，但是通过使用更现代的仪器，可以进一步提高鉴定数量和蛋白质组学分析的覆盖范围，这些仪器的质量准确度和测序速度在过去几年中急剧增加23 ，可以通过采用长时间的液体梯度梯度来覆盖大部分蛋白质组，无需任何预分步骤与高分辨率MS仪器相结合，具有快速排序速度24 。 技术对现有/替代方法的重要意义识别和量化人类心脏瓣膜中的蛋白质（如二尖瓣）的能力是一个重要且具有挑战性的任务，有助于阐明瓣膜疾病中生理/病理过程的机制。定义二尖瓣蛋白质组的变化将大大增加对与组织疾病状态相关的生物过程的性质的理解。 目前关于二尖瓣病理生理学的知识是有限的，通常通过分析涉及特定过程的个体蛋白质，如细胞外基质重塑，止血，炎症或氧化应激25 </s主要通过免疫组织化学在组织水平进行研究。如引言中所预期的，文献中最小的蛋白质组学数据集中在病理性二尖瓣9,10的分析中。 缺乏综合的蛋白质组学研究可归因于细胞外基质蛋白（ 即蛋白聚糖，胶原蛋白和弹性蛋白）高度富含的这种低细胞组织的复杂性。这些蛋白质占总量的约80％，妨碍了低丰度蛋白质26的分析。 因此，为了描述该组织的蛋白质组，需要建立一种有效的提取方案以使蛋白质溶解最大化。该方案允许从1mg组织中提取约50μg蛋白质。与"软"组织相比，这是相对较低的产量作为肝脏（从1mg组织中〜135μg），但对各个样品进行蛋白质分析就足够了。这在定义现象的个体内变异性时尤为重要。 此外，该方法具有与许多分析应用兼容的优点。溶解在所提出的提取缓冲液中的二尖瓣蛋白质可以直接用于基于二维电泳和液相电泳11,12的免疫印迹和蛋白质组学分析，或者在蛋白沉淀后，为了消除缓冲液成分的干扰，可用于其他测定，例如无凝胶质谱法15 。 随着该提取方案的应用，通过鉴定许多细胞内质粒获得正常二尖瓣组织的蛋白质组分的更详尽的表征细胞蛋白。这些蛋白质不仅在细胞外基质中位于胞质溶胶或细胞器中，而且具有不同的分子和生物功能。还鉴定了其他有趣的蛋白质（ 即 CryAB，septin-11，FHL-1和皮肤粘蛋白）。这些蛋白质在二尖瓣中具有未知功能，但其生物学特性表明在瓣膜疾病中可能起作用。 掌握此技术后的未来应用或方向使用该方案，可以将关于蛋白质表达的数据与定量mRNA表达和非定量免疫组织化学分析的数据相关联。事实上，当一起使用这些方法将导致更全面地了解从mRNA到翻译后蛋白质修饰的基础疾病的分子机制。因此，这种方法对于集中在心脏瓣膜病理生理学的研究人员而言是值得关注的。鳍该方案也可应用于与二次阀27非常相似的猪二尖瓣，并用作阀功能评估的实验模型。

最近的证据改变了对mRNA合成后发生的许多调节机制的作用的理解。实际上，翻译，转录后和蛋白水解过程可以调节蛋白丰度和功能。假设转录本水平是蛋白质丰度的主要决定因素，mRNA的浓度代表相应蛋白质的代谢 - 已被部分修订。目前，转录本水平仅部分预测蛋白质丰度，这表明转录后事件发生调节细胞内的蛋白质1,2 。 此外，蛋白质最终决定细胞的功能，因此决定其表型，其可以经受自分泌，旁分泌和内分泌因子的响应的动态变化;血液介体;温度;药物治疗和疾病发展换货。因此，以蛋白质水平为重点的表达分析有助于表征蛋白质组，并且揭示其作为疾病发病机理的一部分发生的关键变化3 。 因此，尽管现有的技术挑战，蛋白质组学所提出的澄清健康和疾病状况的机会也是艰巨的。蛋白质组学可以贡献的特别有希望的研究领域包括:在任何水平（ 即全细胞或组织，亚细胞区室和生物液体）上鉴定改变的蛋白质表达;用于疾病诊断和预后的新型生物标志物的鉴定，验证和验证;并希望能够鉴定可用于治疗目的的新蛋白质靶标，以及药物功效和毒性的评估。 捕捉复杂性蛋白质组是技术挑战。目前的蛋白质组学工具提供了进行大规模，高通量分析来鉴定，定量和验证改变的蛋白质水平的机会。此外，引入分馏和富集技术，旨在避免由最丰富的蛋白质引起的干扰，还通过包括最少丰富的蛋白质来改善蛋白质鉴定。最后，蛋白质组学被补充了翻译后修饰的分析，其逐渐成为蛋白质功能的重要调节剂。 然而，分析中的生物标本中的样品制备和蛋白质回收仍然是蛋白质组学工作流程中的限制步骤，并增加可能陷阱的潜力5 。事实上，在大多数必须优化的分子生物学技术中，第一步是组织匀浆离子和细胞裂解，特别是在不存在扩增方法的低丰度蛋白质的分析过程中。此外，蛋白质的化学性质可以影响其自身的恢复。例如，高疏水性蛋白质的分析是非常具有挑战性的，因为它们在等电聚焦过程中容易沉淀，而跨膜蛋白几乎不溶（参见参考文献5）。此外，组织组成变异性为开发通用提取方法创造了显着的障碍。最后，由于几乎所有临床标本的数量都是有限的，所以必须以最小的样品量使蛋白质制备具有最大的恢复和重现性。 这项工作描述了从正常人心脏二尖瓣进行蛋白质提取的优化方案，这代表了蛋白质组学分析的非常具有挑战性的样品。正常二尖瓣是一种lex结构位于左心房和心脏左心室之间（ 图1 ）。它在控制从心房到脑室的血液流量方面起着重要的作用，防止回流，并确保适当的氧气供应给全身，从而保持足够的心输出量。然而，它通常被认为是一种"无活性"组织，细胞率低，组成少，主要是细胞外基质。这是因为在正常情况下，驻留的瓣膜间质细胞（VIC）具有低蛋白质生物合成速率7的静止表型。 然而，已经证明，在病理状态下，海绵体中的VIC的数量增加并且其蛋白质合成被激活，以及其他功能和表型变化8 。因此，最少的数据可用并不奇怪文献集中于病理性二尖瓣9,10的分析，其中活化的VIC的增加的数量可能解释相对较高数量的鉴定的蛋白质。 总之，本议定书可能通过研究二尖瓣蛋白组分来发展对负责二尖瓣疾病的致病机制的理解。事实上，更深入地了解潜在的病理过程可能有助于改善瓣膜疾病的临床管理，其目前的干预指标主要取决于血液动力学考虑因素。

<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

细胞蛋白质组学的分析可以帮助阐明基于疾病的分子机制，这是由于技术的发展，允许大规模鉴定和定量复制生物系统中存在的蛋白质。从蛋白质组学方法获得的知识有可能导致更好地了解基础疾病的致病机制，允许鉴定新的诊断和预后疾病标志物，以及希望的治疗靶点。然而，心脏二尖瓣代表蛋白质组学分析的非常具有挑战性的样品，因为蛋白多糖和富含胶原蛋白的细胞外基质的细胞数量较少。这使得挑选提取蛋白质进行全球蛋白质组学分析。该工作描述了与随后的蛋白质分析相容的方案，如定量蛋白质组学和免疫印迹。这可以允许数据关联的相关性g蛋白表达与定量mRNA表达和非定量免疫组化分析数据。实际上，这些方法在一起执行时，可以更全面地了解基因疾病的基因分子机制，从mRNA到翻译后蛋白质的修饰。因此，这种方法可以与对心脏瓣膜病理学研究感兴趣的研究者有关。

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

人二尖瓣的蛋白质组成仍然是部分未知的，因为其分析由于细胞数量低，因此通过低蛋白质生物合成而复杂化。这项工作提供了有效提取蛋白质用于二尖瓣蛋白质组分析的方案。

从人体二尖瓣提取蛋白质的优化方案

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

optimized-protocol-for-extraction-proteins-from-human-mitral

Nanyang Technological University

Research

JoVE Journal

Biochemistry

12.8K 次观看.  Centro Cardiologico Monzino IRCCS. 人二尖瓣的蛋白质组成仍然是部分未知的，因为其分析由于细胞数量低，因此通过低蛋白质生物合成而复杂化。这项工作提供了有效提取蛋白质用于二尖瓣蛋白质组分析的方案。 

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

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

科研

生物化学

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

生物素化细胞表面蛋白的纯化<emRigicephalus microplus</em&gt;上皮细胞

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

用于稳定碳氧同位素分析的牙釉质碳酸盐的取样与预处理

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

蛋白质的表征(按尺寸-排除色谱与多角度光散射相结合(SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

优化了炔基脂肪酸类似物的掺入，用于使用点击化学检测脂肪酰化蛋白

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

顺磁弛豫增强，用于检测和表征固有无序蛋白质的自缔合

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center of attention, specifically acidic cannabinoids. The focus is turning toward acidic cannabinoids due to their lack of psychotropic activity. Cannabis plants produce acidic cannabinoids with hemp plants producing low levels of psychotropic cannabinoids. As such, utilization of hemp for acidic cannabinoid extraction would eliminate the need for decarboxylation prior to extraction as a source for the cannabinoids. The use of solvent-based extraction is ideal for obtaining acidic cannabinoids as their solubility in solvents such as supercritical CO2 is limited due to the high pressure and temperature required to reach their solubility constants. An alternative method designed to increase solubility is ultrasonic-assisted extraction. In this protocol, the impact of solvent polarity (acetonitrile 0.46, ethanol 0.65, methanol 0.76, and water 1.00) and concentration (20%, 50%, 70%, 90%, and 100%) on ultrasonic-assisted extraction efficiency has been examined. Results show that water was the least effective and acetonitrile was the most effective solvent examined. Ethanol was further examined since it has the lowest toxicity and is generally regarded as safe (GRAS). Surprisingly, 50% ethanol in water is the most effective ethanol concentration for extracting the highest amount of cannabinoids from hemp. The increase in cannabidiolic acid concentration was 28% when compared to 100% ethanol, and 23% when compared to 100% acetonitrile. While it was determined that 50% ethanol is the most effective concentration for our application, the method has also been demonstrated to be effective with alternative solvents. Consequently, the proposed method is deemed effective and rapid for extracting acidic cannabinoids.

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Ultrasonic-assisted extraction (UAE) increases extraction efficiency of solvents and when applied to Cannabis spp. biomass it reduces the time required for extraction. This decreases the cost and potential cannabinoid loss due to degradation. Additionally, UAE is considered a green method due to low solvent use.

从 大麻 生物质中超声辅助提取大麻二酸

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center ...