作者感谢马蒂亚斯·曼恩博士的鼎力支持和卡塔琳娜Zettl夫人展示的方法。 这项工作是科学进步的支持马克斯 - 普朗克协会。

1。仪表所需<ol><li>用于显微切割和裂解样品制备组织切片<ol><li>加热块设定到99°C或用开水洗澡。 </li><li>激光器的工作microdissector（例如PALM，蔡司，哥廷根，德国）。 </li><li>恒温台式离心机，温度设定为20℃。 </li><li>湿室（箱）与机架的Eppendorf型处理管。 </li><li>孵化器设定为37°C。 </li><li>荧光分光光度计使荧光激发近紫外光与微观尺度的石英比色皿（0.1-0.2毫升）的测量。 </li></ol></li></ol> 2。解决方案<ol><li> “组织裂解液”（TLB）：为0.1M的Tris-HCl，pH值8.0，0.1M DTT，0.5％（重量/体积）聚乙二醇20000和4％SDS中。 </li><li> UA解决方案：8M尿素的0.1M的Tris-HCl pH值为8.5。准备每1样品1毫升UA和IAA的解决方案必须新鲜配制，并在一天之内使用。 </li><li> IAA的解决方案：0.05 M Iodoacetamide在UA。准备每样0.1毫升。 </li><li>内蛋白酶赖氨酸-C原液。 </li><li>胰蛋白酶，原液。 </li><li> “消化缓冲”（DB）：0.05 mol的Tris-HCl pH值为8.5。准备每样0.25毫升。 </li><li> &#39;色氨酸标准溶液&#39;：0.1微克色氨酸1毫升去离子水。 </li><li> “测定缓冲液B&#39;（ABB）：10mM的Tris-盐酸，pH值7.6。 </li><li>原液布里顿和罗宾逊通用缓冲液（BRUB）的。制备含0.1M CH 3 COOH，0.1 MH 3 PO 4和0.1 MH 3 BO 3的溶液，用1M的NaOH调节至所需的pH值（2，4，5，6，和11）。在使用前稀释5倍的去离子水。 </li><li>缓冲液A：1％（V / V）CH 3 COOH在去离子水中。 </li><li>缓冲液B：60％（V / V）CH 3 CN，1％（V / V）CH 3 COOH中的去离子水。 </li></ol> 3。移液器吸头和&#39;StageTip专栏<ol><li>通过堆叠6层Empore，阴膜在共同准备SAX尖柱MMON0.2毫升的枪头。使每个样品2 SAX尖柱。 </li><li>在0.2毫升吸管尖端堆叠3层Empore-C 18的准备“StageTip”。使每个样品6级提示。 </li></ol> 4。组织切片进行显微切割的制备及样品裂解<ol><li>切割用切片机切片（切片的厚度取决于样品。一般显微能够很好地为7-10微米的切片）。 </li><li>照射膜载玻片（MembraneSlide 1.0 PEN）与UV光45分钟。 </li><li>安装在照射到膜幻灯片和干燥的部分，在37℃下2小时。 </li><li> Deparaffinize的安装部分通过在二甲苯中的两个连续孵育2.5分钟和1.5分钟。通过连续孵育在无水乙醇，70％乙醇和水，每1分钟，再水合的部分。 </li><li>染色与Mayer的苏木20秒的部分，用清水冲洗1分钟，并风干。 </li><li>收集细胞populati在使用激光捕获显微切割系统的兴趣。当使用Palm仪（蔡司）收集粘合瓶盖样品（胶粘剂第200章）。 </li><li>吸取TLB中的等分试样通过在盖的显微切割的材料。关闭管和一个短暂离心收集悬浮液。裂解在99℃，搅拌（600转）1小时加热块的显微组织。使用3微升缓冲液为10 NL的显微组织。 </li><li>通过离心澄清粗提物在16000 XG在18°C下10分钟。裂解物可以在-20℃冷冻储存</li></ol> 5。来样加工<ol><li>混合多达50微升的澄清裂解物用200μlUA中的超滤单元（法医30K）和离心机以14,000×g离心，直到小于10微升的溶液将保留在过滤器中。通常这个步骤需要10-15分钟离心。 </li><li>吸取200μl的UA的超滤单元与离心机为5.1。空的收集管。 </li><li>移液管加入50μl的生长素溶液中并在600rpm下在热混合器中混合1分钟并离心在5.1混合。 </li><li>吸取100微升UA的超滤装置和离心机为5.1。两次重复此步骤</li><li>吸取100微升的DB到5.1超滤装置和离心。重复此步骤两次。 </li><li>传送过滤单元，以新的收集管。移液管加入40μlDB用内蛋白酶赖氨酸-C（蛋白酶，以1:100的总蛋白的比例），并混合在600rpm下在热混合器中混合1分钟。在37℃18小时孵育单位在潮湿的腔室中。 </li><li>离心过滤装置在14,000 XG如5.1。 </li><li>吸移管160微升的去离子水和离心机的过滤器单元在5.1。汇集流通（5.7和5.8级）包含由内蛋白酶赖氨酸C.释放肽</li><li>超滤单元转移到新的收集管。加入40微升DB用胰蛋白酶（酶蛋白的比例为1:100），并混合在600rpm下在热混合器中混合1分钟。在37℃下搅拌4小时孵育单位在潮湿的腔室中。重复步骤5.7和5.8收集胰蛋白酶肽。 </li></ol> 6。该肽的定量<ol><li>可以在稀释缓冲液中进行的蛋白质酶解肽含量的测定，因为色氨酸残基以及可访问的溶剂。 </li><li>吸取0.2 ABB毫升到石英比色皿，并记录为“空白”的发射光谱。 </li><li>使用0.1微克的步骤加入TSS的1微升等分到比色皿和温柔的组合，均采用ABB公司准备校准曲线。 </li><li>清洁的试管中。 </li><li>吸取样品并记录光谱（样品）。 </li><li>计算出的蛋白质和肽浓度的 自0.1微克色氨酸对应于约9微克蛋白（基于在b中获得的蛋白质的混合​​物的色氨酸含量的平均值为1.1％Ÿ裂解人体细胞）的蛋白质含量在消化样品或肽含量等于： <img alt="式（1）" fo:content-width="2.787in" fo:src="/files/ftp_upload/50589/50589eq1highres.jpg" src="/files/ftp_upload/50589/50589eq1.jpg" /> 其中，F（标准1）为0.1微克色氨酸标准的荧光。该肽含量允许的消化过程的产量计算，并提供必要的信息，下列肽分离和质谱分析。 </li></ol> 7。该LYS-C和胰蛋白酶肽分馏<ol><li>通过稀释与消化赖氨酸-C和胰蛋白酶有，0.2毫升BRUB pH值11，pH值5的分别获得肽的解决方案。 </li><li>组装SAX尖柱的离心管适配器盖。 </li><li>用0.1ml BRUB，pH值为11的赖氨酸-C pepti分离的含0.1毫升甲醇依次洗涤和平衡的SAX尖柱，0.1毫升1M​​ NaOH和3倍DES和与BRUB，pH值为5的胰蛋白酶肽。溶液通过该柱材料的流动是由离心4000×g下促进。 </li><li>洗涤和平衡了C 18 - &#39;StageTips&#39;随后用0.05毫升甲醇中，然后用0.05ml缓冲液B中，并与0.05毫升缓冲液A的制备6-C 18 - &#39;StageTips&#39;; 4的赖氨酸-分离Ç肽和2的胰蛋白酶肽的分离。 </li><li>组装SAX提示列在C 18 - &#39;StageTip“。加载样品溶液进入平衡SAX提示柱和离心柱于5000×g离心3分钟。 </li><li>平衡的“吸管尖&#39;柱，0.1毫升BRUB的，pH值为11或pH值分别为5到LYC-C和胰蛋白酶样。促进离心5000×g下的流动。 </li><li> SAX的提示栏转移到下一个C 18 - &#39;StageTip“。 </li><li>继续该肽的纯化，用BRUB pH为6，图4和2的赖氨酸C示例，并与BRU乙pH值2与胰蛋白酶肽的样品。对于每一个洗脱步骤使用一个单独的C 18 - &#39;StageTip“。 </li><li>用0.05 ml缓冲液A的&#39;StageTips&#39; -洗了C 18 </li><li>洗脱馏分0.05毫升缓冲液B的成直接用于注射的样品进液相色谱系统装配有质谱仪的小瓶中。 </li></ol>

<ol>
	<li>Nagaraj, N., 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+proteome+and+transcriptome+mapping+of+a+human+cancer+cell+line.">Deep proteome and transcriptome mapping of a human cancer cell line.</a> Molecular systems biology. 7, 548 (2011).</li><li>Beck, M., 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=The+quantitative+proteome+of+a+human+cell+line.">The quantitative proteome of a human cell line.</a> Molecular systems biology. 7, 549 (2011).</li><li>Wisniewski, J. 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R., 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=Universal+sample+preparation+method+for+proteome+analysis.">Universal sample preparation method for proteome analysis.</a> Nature. 6 (5), 359-362 (2009).</li><li>Wisniewski, J. R., Ostasiewicz, P., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+recovery+FASP+applied+to+the+proteomic+analysis+of+microdissected+formalin+fixed+paraffin+embedded+cancer+tissues+retrieves+known+colon+cancer+markers.">High recovery FASP applied to the proteomic analysis of microdissected formalin fixed paraffin embedded cancer tissues retrieves known colon cancer markers.</a> Journal of proteome research. 10 (7), 3040-3049 (2011).</li><li>Wisniewski, J. R., 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=Extensive+quantitative+remodeling+of+the+proteome+between+normal+colon+tissue+and+adenocarcinoma.">Extensive quantitative remodeling of the proteome between normal colon tissue and adenocarcinoma.</a> Mol. Syst. Biol. 8, 611 (2012).</li><li>Wisniewski, J. R., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consecutive+proteolytic+digestion+in+an+enzyme+reactor+increases+depth+of+proteomic+and+phosphoproteomic+analysis.">Consecutive proteolytic digestion in an enzyme reactor increases depth of proteomic and phosphoproteomic analysis.</a> Anal. Chem. 84 (6), 2631-2637 (2012).</li><li>Wisniewski, J. 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M., 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=Impact+of+pre-analytical+factors+on+the+proteomic+analysis+of+formalin-fixed+paraffin-embedded+tissue.">Impact of pre-analytical factors on the proteomic analysis of formalin-fixed paraffin-embedded tissue.</a> Proteomics Clin. Appl. , (2012).</li></ol>

作者有没有透露。

通过蛋白质组学技术研究FFPE材料的深度与核酸测序和基因芯片技术相媲美的能力，在打开的生物标志物和药物靶标发现新的前景。所描述的协议使表征和的规模为10,000蛋白质显微细胞群体的蛋白质组定量。当使用较少的显微切割材料的较小的数据集可以被生成，但也许在许多情况下，这些也可以提供有价值的临床数据。因此，无论试样可直接在MED-FASP程序后进行分析，或者可以在更少的馏分分隔。因为FASP过程是与任何类型的蛋白酶相容，其它酶或它们的组合可用于从蛋白质消化6。 将FFPE材料的质量似乎是分析中最关键的问题。我们经历了同样的起源固定的组织，但来自哪里不同耳鼻喉科诊所有不同的特性。从一个源采用组织，我们能够在高产量，产生肽，而来自另一家诊所类似的材料几乎是无用的。它很可能是所施加的固定和嵌入程序，以及存储条件是影响临床材料12的性能的主要因素。因此，建议启动一个大项目之前测试几个样品的性质。 许多出版物报道，在过去使用的FFPE材料制成。然而，在这些研究中所发现的蛋白质的数量没有超过超过1000-2000蛋白4。考虑到包括超过10,000的蛋白质人体细胞特异性蛋白质组的规模，这样的研究能够只提供一个非常肤浅的画面，几乎仅限于参与管家的功能非常丰富的蛋白质。我们的协议使临床或生物材料F的高效分离ROM保存的组织，并为裂解，蛋白加工和肽预分馏优化。因此我们的样品制备工作流程中，耦合到高端的质谱时，允许见解一个几乎完整的蛋白质组。显着的，这有可能在分钟样品量要求。 使用保存的组织的主要优点是它们的相对广泛的可用性。 FFPE样品封存多年，甚至几十年，有时认为是没用的，现在，多亏了蛋白质组学技术，对出现临床研究作为非常有价值的材料。而许多疾病都可以使用FFPE材料的新鲜或冷冻的材料进行调查研究，似乎是研究罕见疾病12特别重要的，是收集的一组样本的代表性，通常需要很长的时间。

固定用多聚甲醛和在石蜡（FFPE）的嵌入是用于保存和临床组织材料病理形态学研究的标准方法。保留的组织的显微镜可以识别疾病相关的形态学变化，并检测发生疾病相关抗原和得分。自FFPE样品通常只有一小部分被用来在这些分析中，大量的临床材料保持封存，可以在其他类型的研究中被利用。 在最近的十年中，生命科学研究已加强了功能强大的蛋白质组学技术。这项技术可以识别成千上万的蛋白质在复杂的蛋白质混合物和定量。该技术的一个重要特征是，所需要的材料只有微量的大规模分析。最近的蛋白质组学研究表明，蛋白可以研究的规模为近小样图勒特蛋白质组1，2。这种类型的调查能够在细胞的组合物和鉴定在异常水平的疾病发生的蛋白质的基团的全系统的见解。而这些研究需要大量的质谱能力，最近的工作表明，识别相似程度可以在测量3的单日实现。 相对低的样品量的要求，保留的临床样本的广泛可用性诱惑我们和其他人开发的方法使蛋白质勘探FFPE材料（综述见4）。考虑下，我们开发了一个协议，允许蛋白质从固定组织5定量提取热处理优势福尔马林固定的可逆性。我们已经表明，在固定过程保留不仅蛋白质，而且至少在某些翻译后修饰。 Phosph过磷酸化和N-糖基化可以使用FFPE样品5，但是一个前提条件，因为这是一个彻头彻尾的控制组织收集，存储和固定的条件进行研究。接下来，我们优化的方法中，固定组织的激光捕获显微切割和蛋白质组学基于反应器样品处理6,7。对结肠直肠癌的大规模研究允许的7,500蛋白质从临床资料8显微切割定量分析。最后，我们通过应用连续，两步蛋白质的消化协议9显微提高材料的探索之路。相比于使用单一酶共同消化策略，此过程能够产生更多的序列独特的肽，并因此导致更高的深度的蛋白质鉴定。允许显微人类结肠腺瘤的蛋白质组分析来分析每个样品3 9,500蛋白质的深度。在这种螺柱Ý我们映射每个样品55000的肽允许标识88-89％的蛋白质具有至少2个肽。在这里，我们提出了一个优化的协议，用于样品制备从FFPE材料LC-MS/MS分析。这个协议包括在反应器中的格式（FASP）制备组织切片进行显微解剖，分离的细胞的裂解，和样品处理6。然后，我们描述了一个荧光分光光度测定肽的产量，与最佳的肽段鉴定高度重视任务的质谱。最后，我们展示了基于 - 肽分馏分离技术强阴离子交换（SAX）。在这种方法既肽分离和最后的清理工作采用吸管尖端微柱10，11。这一步是可选的，但建议在其中超过几微克肽可以从单个样品得到的研究。该SAX-分馏可以大幅提高的ident的数目和动态范围ifications和延长蛋白质序列覆盖率3，10。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>MembraneSlide 1.0 PEN</td>
			<td>Carl-Zeiss Microscopy GmBH, Goettingen, Germany</td>
			<td>415190-9041-000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Adhesive Cap 200 opaque</td>
			<td>Carl-Zeiss Microscopy GmBH, Goettingen, Germany</td>
			<td>415190-9181-000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mayer's hematoxylin solution </td>
			<td>Sigma-Aldrich St. Louis, MO</td>
			<td>MHS32</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Forensic 30k </td>
			<td>Merck Millipore Darmstadt, Germany</td>
			<td>MRCF0R030 </td>
			<td>(Previously sold as Microcon YM-30) </td>
		 </tr>
		 <tr>
			<td>Empore Anion</td>
			<td>Bioanalytical technologies 3M Company ST. Paul, MN </td>
			<td>2252</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Empore C18</td>
			<td>Bioanalytical technologies 3M Company ST. Paul, MN</td>
			<td>2215</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin </td>
			<td>Promega </td>
			<td>2014-06-27</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Endoproteinase Lys-C</td>
			<td>Wako</td>
			<td>129-02541</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

proteomic sample preparation from formalin fixed and paraffin embedded tissue

保存完好的临床材料是用于人类疾病的蛋白质组学研究的独特来源。在这里，我们描述了一个优化的协议，允许福尔马林大规模定量分析固定，石蜡包埋（FFPE）的组织。该过程包括四个不同的步骤。第一个是从FFPE材料和所关注的细胞的显微切片的制备方法。在第二步骤中分离的细胞溶解，并使用“滤计算机辅助样品制备”（FASP）技术进行处理。在此步骤中，蛋白质被从用于该样品裂解试剂耗尽，被消化中使用内切蛋白酶型lysC和胰蛋白酶两步。 每个消化后，将肽收集在单独的组分和它们的内容是使用一个高度敏感的荧光测量来确定。最后，肽分馏的“吸管尖端”微柱。该LYSC肽被分离成4部分，而胰蛋白酶的peptides被分离成2部分。以这种方式制备的样品允许蛋白质组分析从微量物质到10000的蛋白质的深度。因此，所描述的工作流是一种强大的技术用于研究疾病在一个系统范围内的时尚以及用于识别潜在的生物标志物和药物靶标。

FFPE材料的蛋白质组学分析需要强大的和可重复的方法进行样品制备。在此出版物中，我们展示一个协议，允许细胞群从固定材料有效的隔离和其导致可直接用于LC-MS/MS分析高纯度的肽级分的化学处理。该过程包括四个不同的部分：（1）制备的FFPE样品，其显微切割，（2）裂解使用MED FASP途径的蛋白质的样品和处理，以及（3）定量和（4）的分馏部分的的肽（ 图1）。 在该过程的第一步骤中，FFPE样品被切片用切片机和安装在膜覆盖的幻灯片。以增加组织切片和滑动膜的表面之间的粘附性是必要的照射滑动表面用UV光。为了这个目的，我们使用的UV灯的光集成到一个细胞培养物台上。在此步骤之后的载玻片进行洗涤，允许除去石蜡的样品和补液。再水化材料被染色用苏木的时间很短。这会导致样品的弱染色，但足以为显微样品区域的可视化。在染色过程中，必须通过用过量的水漂洗玻片迅速停止。在可怜的肽的产量抽样调查结果长时间染色。 在接下来的步骤中，干燥段经受显微切割。单个细胞的组织区域的选择取决于调查的类型和总是需要在组织学或病理形态学的具体知识。激光释放出来的材料被收集在样品收集管的粘接表面。由于粘合材料的结合能力是有限的，需要的显微切割的材料从盖传递到吨的样品每3-5毫米2解剖后，他管。这可以通过的组织裂解缓冲液（LTB）的上盖子和管子在离心短的纺丝几微升的移液很容易地完成。后的样品被收集在管的底部，盖子上的显微切割和样品收集可以继续。一旦整个样品收集管，必须额外地塞住用封口膜孵育在水浴中在约99℃下连续搅拌1小时。由于在培养过程中的水凝结在盖，每15分钟的管子必须在短期内离心收集回来的水在管锥。 为了获得肽的组织裂解物使用MED-专上学生资助办法处理。在该方法中，溶解的蛋白质被从洗涤剂和其它低分子量物质，carboamidomethylated在半胱氨酰残基的耗尽，然后连续地消化两种酶DIFfering在他们的裂解特异性：内蛋白酶LYSC和胰蛋白酶。每个消化步骤后的肽被收集到单独的馏分。在这部分的样品制备它继续各自的离心步骤，直到超过95％的最初被装入过滤单元通过该膜的溶液中是非常重要的。分析结肠组织及其癌，我们观察到，该过程产量3-6每100 NL显微切割的材料（ 见表1）的（100微克组织或10 mm 2的10μm的部分的）肽微克。因为从组织中提取的总蛋白为10％左右的组织重量的提取和消化程序一起导致产率的相对于原来新鲜组织30-60％的。这些值反映了该蛋白质的脱水和染色，显微解剖，并保留在超滤单元未消化的样品的所述部分的过程中的损失。因为提取不加工正显微FFPE组织导致蛋白质与肽转化率的75％5很可能在MED-FASP之前的步骤发生的临界样品损失。由该方法得到的肽混合物的一个重要特征是它们的高纯度这有利于进一步分馏肽分馏工艺和质谱鉴定。这个最近已显示通过结肠直肠腺瘤样品3进行分析。在该研究中约40％的MS / MS的事件导致鉴定从FFPE组织的肽，并导致每单LC-MS/MS运行高达17,000肽的映射。仅在冷冻体外培养细胞3的分析更高的识别率已经达到。 在整个过程的最后两个部件的分离的肽进行定量和分级的上移液管尖微柱。由于肽的显微组织中分离出的金额是低10微克它们不能使用常用的染料为基础的蛋白质测定法进行定量。同时在A 280的UV光谱测量在这些浓度是不是由于光散射效应是有用的。与此相反，色氨酸残基的荧光的测量提供用于测定肽含量的可靠方式。 最近，我们已经表明，高达5000的蛋白质从培养细胞可以在“单次”4小时LC-MS/MS分析7确定。但是这种类型的分析应用到本地组织很少可以识别超过3000万人的蛋白质。为了增加分析的MED FASP产生的肽具有被预先分级LC-MS/MS之前的深度。在基于SAX微柱分离是一种简单而高效的分离方法10。它已经在几个蛋白质组学研究包括分析固定的组织5，8，9应用。该SAX-&#39;吸管尖&#39;microcoluMNS，以及C 18 - &#39;StageTip&#39;脱盐柱9顷容易准备。该列由堆叠插头组装，切从SAX或C 18的膜，在200微升移液器吸头11。该SAX-分馏FFPE样品的分析的例子示于表1。 <img alt="图1" src="/files/ftp_upload/50589/50589fig1.jpg" /> 图1。过程概述 。该过程包括四个不同的部分：（1）制备的FFPE样品，其显微切割，（2）裂解使用MED FASP途径的蛋白质的样品和处理，以及（3）定量和（4）的分馏部分的肽。 <table border="1" class="jove_content_small" fo:keep-together.within-page="always"><tbody><tr><td fo:width="1.4in"> 样品 </td><td fo:width="1in"> 样品预分级/消化 </str翁&gt; </td><td fo:width="1in"> 使用质谱仪 </td><td fo:width="1.25in"> 肽产量纳克/ nL的样品（±SD） </td><td fo:width="1.25in"> 独特的蛋白鉴定每一个样本 </td><td fo:width=".75in"> 参考 </td></tr><tr><td> •Adenonocarcinoma •正常结肠黏膜</td><td> SAX /一种酶</td><td>轨道阱，Velos的</td><td> 36±11（N = 8） 30±8（N = 8） </td><td> 5,985±54 5,868±110 </td><td> 8 </td></tr><tr><td> •结肠腺瘤</td><td> SAX /两种酶</td><td> Q Exactive </td><td> 56±6.1（N = 3） </td><td> 9,501±28 </td><td> 3 </td></tr></tbody></table> 表1。的FFPE显微组织的蛋白质组分析的代表性结果。

Watch this Scientific Journal Video about Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue at JoVE.com

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

档案福尔马林固定，石蜡包埋（FFPE）的临床样本进行疾病调查有价值的材料。在这里，我们展示了一个样品制备工作流程允许显微FFPE组织的深入蛋白质组学分析。

修正了福尔马林蛋白质组学样品的制备和石蜡包埋组织

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

proteomic-sample-preparation-from-formalin-fixed-paraffin-embedded

Research

JoVE Journal

Biology

27.4K 次观看.  Max Planck Institute of Biochemistry. 档案福尔马林固定，石蜡包埋（FFPE）的临床样本进行疾病调查有价值的材料。在这里，我们展示了一个样品制备工作流程允许显微FFPE组织的深入蛋白质组学分析。 

视频: Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

免疫组化：石蜡切片从Vector实验室使用Vectastain ABC试剂盒

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

科研

生物学

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

电泳分离蛋白质

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

毒素诱导和蛋白质提取镰刀 SPP。</em蛋白质组学研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

免疫印迹检测样品制备

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

福尔马林固定石蜡包埋的组织核心的制备RNA和DNA提取

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...