This work was funded and facilitated through the GOSomics research initiative by the National Institute for Health Research, Biomedical Research Centre at Great Ormond Street Hospital and the UCL Biological Mass Spectrometry Centre at the UCL Institute of Child Health with kind donations from the Szeban Peto Foundation. The Dementia Research Centre is an Alzheimer's Research UK coordinating centre. The authors acknowledge the support of Alzheimer's Research UK, the NIHR Queen Square Dementia Biomedical Research Unit, UCL/H Biomedical Research Centre, and Leonard Wolfson Experimental Neurology Centre.

注意:此处描述的总体协议的示意图在连接 古尔1中给出用于这种方法的开发所有样品是过剩的临床诊断的样品，并有从伦敦布卢姆斯伯伦理委员会的伦理批准。 <img alt="图1" src="/files/ftp_upload/54541/54541fig1.jpg" /> 图1.示意图龙虎斗创建用于评估靶向CSF的MRM的LC-MS / MS测定。候选标记的肽的整体流程从蛋白质靶选中。通过使用定制合成肽，创建有针对性的LC-MS / MS复用方法。评价后，该测定可用于评估神经变性潜在标志物的功效。 1.肽选择与设计注意:标准标记肽是它必须是唯一的（ 蛋白典型 </STRONG&gt;），并代表蛋白质（quantotypic）的定量丰盈。要确定是否肽是独一无二与否，UNIPROT网站上的&#39;爆炸&#39;的搜索工具（http://www.uniprot.org/blast/）都可以使用。 <ol><li>定义靶蛋白（标记）， 即列表，载脂蛋白E（ 见表2）。 注意:如果标记已经从以前的蛋白质组学分析实验确定了8，然后选择给出了该数据集的最好回应肽。 </li><li>如果此信息不可用，使用开源的网站， 如靶蛋白硅片胰酶消化，使用软件工具，如MS-文摘。 </li><li>选择哪个是胰蛋白酶和不容易受到翻译后修饰的肽。 注:此信息可在网站UNIPROT上www.uniprot.org进行检查。避免LC-MS样品制备过程中易发生化学修饰肽。 </li> &lt;LI&gt;订购选择肽定制合成。 注意:标记肽可以通过各种商业企业可以定制合成。对于ApoE基因被确定用来衡量总ApoE基因水平quantotypic肽是AATVGSLAGQPLQER。用于确定E2和E4变种蛋白典型肽序列是位置158（RLAVYQAGAR和CLAVYQAGAR）和位置112（LGADMEDVCGR和LGADMEDVR）的胰蛋白酶肽，分别。 </li></ol> 2.标准肽的制备注意:为了选择最佳的量化转换，需要优化的矩阵（CSF）的检测。优化复肽的最有效的方式是在已知浓度来创建肽的池。然后这些池可用于方法的开发和标准曲线。 <ol><li>重悬的合成肽（肽细节在表2中给出）至1mg / ml储备根据浓度生产商的说明。默认情况下，如果指令是不可用，在50:50（体积/体积）乙腈（ACN）重悬肽/ H 2 O </li><li>从原料浓度和每种肽的池1000皮摩尔成低结合的离心管制备该肽的1:10稀释液。干倒在一个速度真空浓缩器在-20°C的最终游泳池和商店。准备几个游泳池供日后使用。 </li><li>分装100微升CSF进入低结合管。冻干脑脊液。 </li><li>重悬在消化缓冲液（100mM的Tris盐酸，pH值7.8，6M尿素，2M硫脲，2％ASB14），得到10和11皮摩尔/微升的浓度汇集1000皮摩尔肽的等分试样。 </li><li>穗汇集肽进入CSF的冻干100微升等分试样在0，1，2，5，10和15时的浓度。添加20ng的完整无关蛋白如酵母烯醇化酶作为内标，并控制为TR的消化效率ypsin。 </li><li>加满消化缓冲液的20微升的最终量CSF等分。涡流。 </li><li>添加二硫苏糖醇的1.5微升（30毫克在1ml的100mM的Tris盐酸，pH值7.8）和在室温下振摇1小时。 </li><li>加入3微升碘乙酰胺（35毫克在1ml的100mM的Tris盐酸，pH值7.8）和在室温下振摇在黑暗中45分钟。 </li><li>加入165微升的DDH 2 O的</li><li>添加0.1微克10微升/微升重新悬浮于50mM碳酸氢铵缓冲液，pH 7.8测序级改良的胰蛋白酶溶液。孵育在37℃CO / N的水浴并通过冷冻样品停止消化。储存在-20°C消化，直到准备分析。 </li></ol> 3. MS肽检测的优化<ol><li>复制并粘贴肽的序列被优化，到适当的软件， 如，天际线9。点击肽序列获得的信息预计前体离子的质量和产品的离子。 </li><li>从稀释股权集中肽（参见2.1节），进一步为1纳克/毫升的浓度MS方法开发。 </li><li>直接注入质谱仪中的肽在优化流动速率（通常为0.1 - 0.8毫升/分钟），并用0 - 5％碰撞能量。 </li><li>获得MS谱以鉴定实验多电荷前体离子8。选择给出最强度（doubly-或三重充电前体离子被决定）的前体离子（M / Z）。 </li><li> Reinfuse肽和应用碰撞能量片段通过碰撞诱导解离（CID）的肽。优化锥体和碰撞能量，以获得最佳的碎片图案（碎片离子优选大于母体离子大的碎片离子单独或双电荷，和质量，宜）。 </li><li>验证实验获得的过渡相匹配，在硅片中产生的过渡&lt;/ em&gt;的（如在步骤3.1中所述）。保存使用每个母离子的至少2个最激烈的过渡中的MRM方法文件的转换列表。包括2个转换:1进行定量和1个在最终检测确认。 注:某些MS厂家有一个功能（ 即 ，沃特世智能启动），用于自动MRM或SRM分析优化。如果可能的话，在50注入与组合的移动相肽 - 70％ACN的0.1％FA。 </li></ol> 4. LC-MRM方法开发注意:由超性能耦合到三重四极质谱仪液相色谱（UPLC）系统分析合成肽的混合物。确保源是干净的。溶剂A是的DDH 2 0 0.1％FA;溶剂B是ACN用0.1％的FA。 <ol><li>使用配有填充用C-18相（直径1.6微米，90埃的孔，2.1毫米×50毫米长），并连接到同一相的预柱一UPLC色谱柱的LC-MS系统。&lt;/ LI&gt; <li>在冰上，离心机除霜CSF消化以16,000 g下10分钟，并转移60微升到300微升玻璃插入小瓶中并在-20存储靠背℃。 </li><li>从注射标准曲线点使用10分钟1浓度最高- 40％ACN线性渐变（见渐变设置表1） </li><li>打开生成的色谱图和笔记保留时间和顶部的两个最激烈的（定量）每肽转换与在步骤3中创建的所有过渡。 </li><li>基于该信息，更新定时信道的10分钟MRM方法（在步骤3.6中创建）来衡量肽（参见图2的例子）。维持灵敏度，保持与每峰大于8分每个信道和停留时间小于0.01秒，对于每种肽的至少一种过渡更大。 </li><li>包括在MRM方法"溶剂延迟":一个是在年初到第一峰洗脱前10秒，另在该方法中，最后的峰值洗脱后20秒的结束。通过在MS方法文件方法事件中选择"溶剂延迟"做到这一点。 </li><li>通过定时MRM方法运行标准曲线，并确保有与转换（步骤3.6生成的）无干扰的非特异性峰通过检查线性。 </li><li>确定该肽是通过运行通过该方法非加标池控制和疾病脑脊液检测的。 </li><li>删除低于检测从检测限的肽。 </li></ol><img alt="图2" src="/files/ftp_upload/54541/54541fig2.jpg" /> 肽转变图2.动态MRM MS方法的实施例。定时通道可以按既定的保留时间进行分组。使MRMS被纳入一个定时方式作为所选择的标记肽从层析柱洗脱出来，最小化TRA的数目在一段时间内nsitions，提高了检测的灵敏度。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54541/54541fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 5.加入内标准注意:如前面10所描述的，稳定的同位素标记的内标可以包含在测定中。由于这些标准的费用，这是应先评估在基质中的肽。 <ol><li>确定用于检测CSF中的优化提供了理想的肽:选择，这是最经常的肽，具有最高的强度和无干扰峰。 </li><li>相应的肽的设计，以包括重13 C 15 N氨基酸的标签，达相对于内源性肽的至少6通过增加肽的质量。此外，添加的4标记 - 重打气的6个附加氨基酸N-或C-末端潮来控制胰蛋白酶消化。 </li><li>稀释消化缓冲液的稳定同位素标记的内部标准（见步骤2.4）。确定将在脑脊液通过在取决于开发期间先前观察到的丰度各级扣球被掺入稳定同位素标记的内标物的理想的量。旨在实现约为1:内源性肽1比稳定同位素标记的标准。 注:稳定同位素标记的内标可通过各种商业公司进行合成。 </li></ol>脑脊液患者样品的6 LC-MRM测定<ol><li>穗优化的稳定同位素标记的标准量为100微升脑脊液并冷冻干燥该混合物。 </li><li>重悬CSF的20微升消化缓冲液和作为分2.7描述进行消化 - 2.10。 </li><li>分析使用步骤4中开发的LC-MRM方法的样品。 </li><li>对于定量分析，运行标准PEP潮汐在标准曲线的0 -15皮摩尔浓度。请参阅步骤2.5标准曲线的准备。 </li></ol> 7.数据分析注:定量数据是基于基线峰内部标准（重标记的肽）的强度比。对于SRM / MRM数据分析的详细信息，前面所述10。比率数据然后可以在标准曲线被用来确定绝对水平或由重标记的肽的加入浓度计算它们。 <ol><li>使用质谱仪制造商的标准软件10分析LC-MRM数据。或者，使用天际线软件分析定量MRM数据10。 </li><li>检查通过检查内部标准的响应运行的灵敏度等的尖刺酵母烯醇化酶或在每次运行稳定的同位素标记的标准。确保变化（CV）的系数不大于25％。 </li><li>手动查看数据标注，以确保准确性。每种肽和比率分析为合适的稳定同位素标记的内标， 即，如果有的话使用的肽的稳定同位素标记的版本。 </li><li>以得到每100微升脑脊液皮摩尔的绝对值，通过分别在同一时间运行适当的标准曲线运行比率数据。 </li><li>计算的变化（CV）的系数。的CV对于每种肽应低于25％和15％以下为高丰度的肽。 注:每100微升CSF值绝对值皮摩尔可在随后的下游统计分析使用。 </li></ol> 8.载脂蛋白E亚型状态注意:要确定的ApoE同种型状态，对应的肽的存在可以通过确定每个同种型的存在下进行。 <ol><li>考虑所述ApoE在100μl&gt; 1000信号的脑脊液阈值底噪为阳性为该肽。参见图5为所需要的肽/缺席确定患者的同种型地位。通过计算各亚型的％，总计ApoE基因表达确定等位基因表达。 </li></ol>

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The authors have nothing to disclose.

如同所有的基于MS测定法，在该方法的关键步骤是内部标准的适当和准确的量的测定。如果使用绝对定量，则正确的量的标准曲线加标肽的也是关键的。 我们的测定法不需要CSF或使用任何类型的MS分析之前清理或脱盐步骤的析出 - 它是一个完全一锅反应方法。由于CSF的体积小和它的有限复杂性（相比于等离子体），已经发现，这些步骤可以从最终的协议被去除，从而简化了测定，使之更适合于翻译成诊断设置。预柱的主要分析柱和在MS方法的溶剂延迟的夹杂物，似乎足以维持该MS中灵敏度运行超过500个样本。作为所述检测UPLC runni纳克时间短，以明确地确定在脑脊液正确峰消化矩阵是重要的。 这只能通过使用加标合成的肽的和标准曲线来实现。如果存在匹配于正确色谱峰的肽的不确定性则可能运行通过多个过渡样本和与合成标准检查跃迁强度图案是有用的。所有的测定法含有至少2肽转变到确认该肽的身份。 该测定法已被开发用于在高达100微升脑脊液检测标记。痴呆的一些其它标记，可能需要CSF的较大体积。该测定的另一个限制是蛋白质表达的动态范围的问题。一些丰富标记需要在一个较小的量的质谱仪被注入，同时一些低丰度的标记需要较大的注射体积。因此，SAMPLE可需要被注射两次。 该技术的意义在于复用能力和3级的鉴定过的抗体（保留时间，前体和产物M / Z）的提高的特异性。从临床翻译的角度来看，最大的优点是节省了抗体的潜在的成本，虽然是必需的质谱设备和受过训练的人员的初始费用。另一资产是其中该方法可以被翻译到三重四极杆为基础的系统的速度。这显著加快相比，所有基于免疫的技术，翻译和验证化验的能力。最后，这个平台和专业技术已常规用于临床检测小分子，很多大型医疗中心已经在这个地方的基础设施。在神经变性的领域有很多焦点并需要在CSF和血清新生物标志物。这种方法提供了PLATForm的用于高通量测定法，其中未来的标记物可以被添加（和删除）进行评估疗效等 。该技术具有进一步的应用已经被描述2乃至血斑其他组织中的ApoE同种型鉴定，如血浆。

神经变性疾病如阿尔茨海默氏病，路易体痴呆和帕金森氏病，的生长影响成为许多国家1中的社会经济问题。有在可用于识别和在疾病的早期阶段进行分类的患者，并监测任何潜在的新的治疗方法的其他的生物标记物需要。这种方法的总体目标是建立一个通用的管道用于验证神经退行性疾病的潜在CSF标记的精简，经济和快捷的方式。基本原理是利用目标蛋白质组或肽MRM LC-MS / MS作为一种容易可修正方法从实验中发现多个评估潜在的蛋白生物标志物。这些可以在一个快速色谱分离（&lt;10分钟），并评估被进一步复用。在这多重屏幕神经退化生物标志物，我们包括已知的老年痴呆症的危险因素载脂蛋白E4亚型（ApoE4的），所以我们CAN按，消除单独的基因分型测试2需要同时确定其状态和表现水平。 LC MS / MS被常规用作首选优于其它方法如ELISA或放射免疫测定（RIA）准确定量的小分子的方法。这种转变在使用质谱技术来分析蛋白质，已通过与基于免疫的技术有关的问题，主要推动。这些包括交特异性，批次变异，有限的货架寿命和成本高。因此，有针对性的蛋白质组学研究正在迅速成为越来越多的替代基于抗体的方法，如蛋白印迹，RIA和ELISA。然而，多路传输的许多标记到一个测定的能力，是该技术通过基于免疫方法3的主要优势。该技术适用于多种组织中，并已作为许多蛋白质组学研究，包括血浆4和尿5,6-验证策略。 在TEchnique可以应用到具有在使用三重四极杆质谱仪的访问和专业知识的任何实验室。多肽设计与越来越多地使用开源数据库的比较简单。定制多肽合成激烈的市场竞争，使他们更加低廉。重肽，然而，是昂贵的，因此，标记应在一个小的队列移动到较大规模之前进行评估。有越来越多的潜力，以在临床诊断设置中所使用的技术，与具有可以容易地适于运行靶向蛋白质组学测定三重四极杆的平台最大医院。该方法的一种这样的应用，使得它进入常规诊断设置，是其最近应用到新生儿血斑筛查镰状细胞贫血症7。

<table><tbody><tr><td>Acetonitrile (ACN), LC-MS grade&amp;nbsp;</td><td>Fisher</td><td>A955-1</td><td></td></tr><tr><td>Formic acid, LC-MS grade,</td><td>Fisher</td><td>A117-50</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma&amp;nbsp;</td><td>D5545 - 5 g</td><td></td></tr><tr><td>Hydrochloric acid, 37% w/w</td><td>VWR&amp;nbsp;</td><td>BDH3028 - 2.5 LG</td><td></td></tr><tr><td>Iodoacetamide&amp;nbsp;&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>I1149 - 5 g</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Fisher</td><td>S318-500</td><td></td></tr><tr><td>Trypsin, sequencing grade, modified</td><td>Promega</td><td>V5113</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA), LC-MS grade</td><td>Fisher</td><td>A116-50</td><td></td></tr><tr><td>Urea</td><td>Sigma</td><td>U0631- 500 g</td><td></td></tr><tr><td>Water, LC-MS ULTRA Chromasolv</td><td>Fluka</td><td>14263</td><td></td></tr><tr><td>Custom synthesised peptides desalted 1-4 mg</td><td>Genscript</td><td>custom</td><td></td></tr><tr><td>heavy labeled amino acid [&lt;sup&gt;13&lt;/sup&gt;C; &lt;sup&gt;15&lt;/sup&gt;N] custom peptides</td><td>Genscript</td><td>custom</td><td></td></tr><tr><td>ASB 14</td><td>Merck Millipore</td><td>182750 - 25 g</td><td></td></tr><tr><td>Thiourea</td><td>Sigma</td><td>T7875 - 500 g</td><td></td></tr><tr><td>Tris base</td><td>Sigma</td><td>T6066</td><td></td></tr><tr><td>VanGuard precolumn</td><td>Waters</td><td>186007125</td><td></td></tr><tr><td>Cortecs UPLC C18 + 1.6 &amp;#956;m&amp;nbsp; 2.1 x 50 mm column</td><td>Waters</td><td>186007114</td><td></td></tr><tr><td>Yeast Enolase&amp;nbsp;</td><td>Sigma</td><td>E6126</td><td></td></tr><tr><td>300 &amp;#956;l clear screw top glass vials</td><td>Fisher scientific</td><td>03-FISV</td><td></td></tr><tr><td>Y slit screw caps&amp;nbsp;</td><td>Fisher scientific</td><td>9SCK-(B)-ST1X</td><td></td></tr><tr><td>Freeze dryer</td><td>Edwards Mudulyo&amp;nbsp;</td><td>Mudulyo system</td><td></td></tr><tr><td>Concentrator/Speed vaccum</td><td>Eppendof&amp;nbsp;</td><td>concentrator&amp;nbsp;plus 5301</td><td></td></tr><tr><td>Xevo -TQ-S mass spectrometer</td><td>Waters</td><td></td><td></td></tr><tr><td>Acquity UPLC system</td><td>Waters</td><td></td><td></td></tr></tbody></table>

a high throughput, multiplexed and targeted proteomic csf assay to quantify neurodegenerative biomarkers and apolipoprotein e isoforms status

许多神经退行性疾病仍缺乏有效的治疗方法 。识别和这些疾病分类可靠的生物标志物将在以后的新型疗法的发展具有重要意义。通常，潜在的新的生物标志物不让它进入临床，由于其发展和成本高的限制。然而，使用多反应监测液相色谱 - 串联目标蛋白质组/质谱法（MRM的LC-MS / MS），特别是使用三重四极杆质谱仪，是一种方法，可用于快速评估和验证为临床翻译成诊断实验室的生物标志物。传统上，这平台已经广泛地用于临床实验室的小分子的测定，但它是分析蛋白质的潜力，这使得它有吸引力的替代ELISA（酶联免疫吸附吸附法）为基础的方法。我们在这里描述蛋白质组学如何定向可用于测量痴呆复用标记物，其中包括已知的危险因素载脂蛋白E亚型4（个ApoE4）的检测和定量。 为了使该试验适用于翻译，它的设计是快速，简便，高特异性和成本效益。为了实现这一目标，在化验的发展每一步都必须为他们分析个人的蛋白质和组织进行优化。该方法描述了一个典型的工作流程，包括各种提示和技巧，制定有针对性的蛋白质组学MRM LC-MS / MS翻译。 该方法的发展，使用胰蛋白酶quantotypic肽，其校准的MS检测的定制合成版本优化，然后掺入CSF的，以确定在MS分析之前的色谱分离的内源性肽的正确鉴定。为了实现ABSOLU德定量，稳定的同位素标记的内标版本的肽的短的氨基酸序列的标签和含有胰蛋白酶切割位点，包括在测定中。

使用上述的方法中，由来自54蛋白74肽的高通量10分钟多重测定的开发，对于神经变性疾病阿尔茨海默氏病和路易小体痴呆（LBD）8。 图3的标志物的测定显示出版多路复用色谱先前8从测定中显著肽标记。包括在测定及其数量的过渡肽在表2中给出。由这种方法所产生的数据提供了标准化的比率相关的稳定同位素标记的内标。这些值然后可通过标准曲线付诸确定绝对皮摩尔/100μl的CSF浓度。这些值然后可统计学分析临床样品中的变化。 如前所述8中 ，74 PE的定量包括在此CSF检测ptides透露，这些标记的25个老年痴呆症患者的脑脊液显著改变。为了说明该测定的效力，从先前的结果描述性痴呆标记亲食欲素和YKL几丁质酶3样蛋白（YKL-40）11,12给出在图4中该集成的ApoE测定标识的所述ApoE同种型/等位基因状态病人也是如此。载脂蛋白E4为阿尔茨海默氏病的已知风险因素，因此整合到这一点测定会提供有价值的信息。所述ApoE同种型的检测在图5中进行说明，并基于该检测为同种型的氨基酸变化相应肽的E2（R158C）和E4（C112R）。 图5A示出预期的每个同种型的组合的峰图案和图5B示出了具有上的患者样品的测定中测试一个CSF的结果。 图3. 代表叠加的MRM色谱图。从海伍德等人的神经退行性疾病路易体痴呆和阿尔茨海默氏病8显示在10分钟的LC梯度8生物标志物显著再版。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54541/54541fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54541/54541fig4.jpg" /> 图4. 实施例的数据。从海沃德等重印8图说明如何描述的MRM的LC-MS / MS方法能够可靠定量和从控制判别已知神经变性标记如YKL几丁质酶3样蛋白（YKL-40）11 ，12和AD标记镨<html>邻食欲素13。 AD =阿尔茨海默氏病，LBD =路易体痴呆和PD =帕金森氏病。数据先前公布。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54541/54541fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54541/54541fig5.jpg" /> 图 5. 如何病人的载脂蛋白E基因亚型状态才能确定。A.表示肽覆盖中性（E3A）为E4和158位存在的112个氨基酸序列LGADMEDVCGR或LGADMEDVR检测RLAVYQAGAR中性（E3B）或CLAVYQAGAR用于从所述ApoE序列的E2亚型。B.肽示于左侧面板。在脑脊液检测的肽的不同组合可以指示所述ApoE同种型地位。文件/ ftp_upload / 54541 / 54541fig5large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <table border="0" cellpadding="0" cellspacing="0" width="372"><colgroup><col /><col /><col span="3" /></colgroup><tbody><tr height="22"><td height="22" style="height:22px;width:64px;">时间</td><td style="width:116px;">流量（毫升/分钟） </td><td style="width:64px;"> ％ 一个</td><td style="width:64px;"> ％B </td><td style="width:64px;">曲线</td></tr><tr height="22"><td height="22" style="height:22px;width:64px;">初始</td><td style="width:116px;"> 0.8 </td><td style="width:64px;"> 97 </td><td style="width:64px;"> 3 </td><td style="width:64px;">初始</td></tr><tr height="22"><td height="22" style="height:22px;width:64px;"> 0.2 </td><td style="width:116px;"> 0.8 </td><td style="width:64px;"> 97 </td><td style="width:64px;"> 3 </td><td style="width:64px;"> 6 </td></tr><tr height="22"> &lt;TD高度="22"的风格="高度:的22px;宽度:64PX;"&gt; 7 </td><td style="width:116px;"> 0.8 </td><td style="width:64px;"> 60 </td><td style="width:64px;"> 40 </td><td style="width:64px;"> 6 </td></tr><tr height="22"><td height="22" style="height:22px;width:64px;"> 7.01 </td><td style="width:116px;"> 0.8 </td><td style="width:64px;"> 0.1 </td><td style="width:64px;"> 99.9 </td><td style="width:64px;"> 6 </td></tr><tr height="22"><td height="22" style="height:22px;width:64px;"> 8 </td><td style="width:116px;"> 0.8 </td><td style="width:64px;"> 0.1 </td><td style="width:64px;"> 99.9 </td><td style="width:64px;"> 6 </td></tr><tr height="22"><td height="22" style="height:22px;width:64px;"> 8.01 </td><td style="width:116px;"> 0.8 </td><td style="width:64px;"> 97 </td><td style="width:64px;"> 3 </td><td style="width:64px;"> 1 </td></tr><tr height="22"><td height="22" style="height:22px;width:64px;"> 10 </td><td style="width:116px;"> 0。8 </td><td style="width:64px;"> 97 </td><td style="width:64px;"> 3 </td><td style="width:64px;"> 1 </td></tr></tbody></table> 表1. UPLC梯度设置一个10分钟的方法 。 A = 2的DDH 0 0.1％FA，B = ACN，0.1％FA 表2肽中包含的脑脊液的MRM的LC MS / MS分析。显示的是所有的方法中使用的包含肽，其描述和公布8。是否该标记物在100μl脑脊液可靠地检测指示被指示。标记的大胆转变是用于定量数据的人。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54541/Table_2.xlsx" target="_blank">请点击此处下载此表。</a>

Watch this Scientific Journal Video about A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status at JoVE.com

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

我们描述了一种高通量，多重，和有针对性的蛋白质组学脑脊液（CSF）试验与临床翻译潜在发展。该试验可定量潜在标记和危险因素的神经变性，如载脂蛋白E的变体（E2，E3和E4），并测量它们的等位基因的表达。

高通量，复用和有针对性的蛋白质组学分析脑脊液量化神经退行性生物标志物和载脂蛋白E亚型状态

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

a-high-throughput-multiplexed-targeted-proteomic-csf-assay-to

Universidad San Sebastian

Research

JoVE Journal

Medicine

7.7K 次观看.  University College London. 我们描述了一种高通量，多重，和有针对性的蛋白质组学脑脊液（CSF）试验与临床翻译潜在发展。该试验可定量潜在标记和危险因素的神经变性，如载脂蛋白E的变体（E2，E3和E4），并测量它们的等位基因的表达。 

视频: A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. A&beta; is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into &beta;-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble A&beta; multimers are more toxic than fibrillar A&beta; amyloid2. The identification and quantification of low- and high-molecular weight multimeric A&beta; species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring A&beta; multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with A&beta;-specific antibodies. However, the separation and detection of multimeric A&beta; requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the A&beta; peptide. Using this protocol, we consistently detect A&beta; monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the A&beta; peptide. Using this protocol, we consistently detect monomeric and multimeric A&beta; in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot检测人脑皮层组织中的Aβ多聚匀浆使用抗原表位检索

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

科研

神经科学

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

在神经退行性疾病的高内涵筛选

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

医学

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

一个串行收集脑脊液从池鼠标麦格纳技术

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

生物学

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

共识脑源性蛋白，人类和小鼠脑蛋白质组使用两个2D-DIGE和Mini双向电泳免疫印迹法研究提取协议

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step.
Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers.
Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution.
Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.

A method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF) is presented. By employing molecular weight cut-off filtration, chromatographic pre-fractionation, mass spectrometric analysis and a subsequent combination of peptide identification strategies, it was possible to expand the known CSF peptidome nearly ten-fold compared to previous studies.

人脑脊液 Endopeptidomic 分析的样品制备

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

Absolute Quantification of A&#946;1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the diagnosis of AD is based on cognitive tests and mental state exams, but the peptide amyloid-beta (A&#946;) in cerebrospinal fluid (CSF) is increasingly used in clinical trials and settings. As for most protein and peptide biomarkers, quantification is performed using antibody-based techniques, such as enzyme-linked immunosorbent assay (ELISA). However, intra- and inter-laboratory variability in these assays hamper its use as a diagnostic marker in clinical routine.
An antibody-independent Reference Measurement Procedure (RMP) was developed based on solid-phase extraction (SPE) and liquid chromatography (LC)-tandem mass spectrometry (MS/MS), where stable, isotope-labeled A&#946; peptides were used as internal standards, enabling absolute quantification. A high-resolution quadrupole-orbitrap hybrid instrument was used for the measurements. The method allows for the quantification of CSF A&#946;1-42 between 150-4,000 pg/mL.

A reference measurement procedure for the absolute quantification of A&#946;1-42 in human CSF based on solid-phase extraction and liquid chromatography tandem mass spectrometry is described.

Aβ的绝对定量<sub&gt; 1-42</sub&gt;在CSF使用质谱参考测量程序

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the ...

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%&#8211;20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

使用毛细血管电泳免疫测定来寻找人类血小板肌萎缩性侧索硬化的潜在生物标志物

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

来自大脑的淀粉样原纤维核心的生化纯化和蛋白质组学表征

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...