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="http://ecsource.jove.com/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>

<ol>
	<li>Prince, P. M., Guerchet, D. M., Prina, D. 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=.">.</a> Policy Brief: The Global Impact of Dementia 2013-2050. , (2013).</li><li>Hirtz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+new+quantitative+mass+spectrometry+and+semi-automatic+isofocusing+methods+for+the+determination+of+Apolipoprotein+E+typing.">Development of new quantitative mass spectrometry and semi-automatic isofocusing methods for the determination of Apolipoprotein E typing.</a> Clin Chim Acta. 454, 33-38 (2015).</li><li>Marx, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+proteomics.">Targeted proteomics.</a> Nat Methods. 10 (1), 19-22 (2013).</li><li>Heywood, W., 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+development+of+a+peptide+SRM-based+tandem+mass+spectrometry+assay+for+prenatal+screening+of+Down+syndrome.">The development of a peptide SRM-based tandem mass spectrometry assay for prenatal screening of Down syndrome.</a> J Proteomics. 75 (11), 3248-3257 (2012).</li><li>Heywood, W. E., 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=Proteomic+Discovery+and+Development+of+a+Multiplexed+Targeted+MRM-LC-MS/MS+Assay+for+Urine+Biomarkers+of+Extracellular+Matrix+Disruption+in+Mucopolysaccharidoses+I,+II,+and+VI.">Proteomic Discovery and Development of a Multiplexed Targeted MRM-LC-MS/MS Assay for Urine Biomarkers of Extracellular Matrix Disruption in Mucopolysaccharidoses I, II, and VI.</a> Anal Chem. , (2015).</li><li>Manwaring, V., 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+identification+of+new+biomarkers+for+identifying+and+monitoring+kidney+disease+and+their+translation+into+a+rapid+mass+spectrometry-based+test:+evidence+of+presymptomatic+kidney+disease+in+pediatric+Fabry+and+type-I+diabetic+patients.">The identification of new biomarkers for identifying and monitoring kidney disease and their translation into a rapid mass spectrometry-based test: evidence of presymptomatic kidney disease in pediatric Fabry and type-I diabetic patients.</a> J Proteome Res. 12 (5), 2013-2021 (2013).</li><li>Moat, S. J., 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=Newborn+blood+spot+screening+for+sickle+cell+disease+by+using+tandem+mass+spectrometry:+implementation+of+a+protocol+to+identify+only+the+disease+states+of+sickle+cell+disease.">Newborn blood spot screening for sickle cell disease by using tandem mass spectrometry: implementation of a protocol to identify only the disease states of sickle cell disease.</a> Clin Chem. 60 (2), 373-380 (2014).</li><li>Heywood, W. E., 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=Identification+of+novel+CSF+biomarkers+for+neurodegeneration+and+their+validation+by+a+high-throughput+multiplexed+targeted+proteomic+assay.">Identification of novel CSF biomarkers for neurodegeneration and their validation by a high-throughput multiplexed targeted proteomic assay.</a> Mol Neurodegener. 10, 64 (2015).</li><li>MacLean, B., 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=Skyline:+an+open+source+document+editor+for+creating+and+analyzing+targeted+proteomics+experiments.">Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.</a> Bioinformatics. 26 (7), 966-968 (2010).</li><li>Manes, N. P., Mann, J. M., Nita-Lazar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+Reaction+Monitoring+Mass+Spectrometry+for+Absolute+Protein+Quantification.">Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification.</a> J Vis Exp. (102), e52959 (2015).</li><li>Craig-Schapiro, 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=YKL-40:+a+novel+prognostic+fluid+biomarker+for+preclinical+Alzheimer's+disease.">YKL-40: a novel prognostic fluid biomarker for preclinical Alzheimer's disease.</a> Biol Psychiatry. 68 (10), 903-912 (2010).</li><li>Perrin, R. J., 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=Identification+and+validation+of+novel+cerebrospinal+fluid+biomarkers+for+staging+early+Alzheimer's+disease.">Identification and validation of novel cerebrospinal fluid biomarkers for staging early Alzheimer's disease.</a> PLoS One. 6 (1), e16032 (2011).</li><li>Liguori, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orexinergic+system+dysregulation,+sleep+impairment,+and+cognitive+decline+in+Alzheimer+disease.">Orexinergic system dysregulation, sleep impairment, and cognitive decline in Alzheimer disease.</a> JAMA Neurol. 71 (12), 1498-1505 (2014).</li></ol>

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?...

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 border="0" cellpadding="0" cellspacing="0" width="542">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Acetonitrile (ACN), LC-MS grade&#160;</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">A955-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formic acid, LC-MS grade,</td>
			<td>Fisher</td>
			<td>A117-50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td>Sigma&#160;</td>
			<td style="width:119px;">D5545-5G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid, 37% w/w</td>
			<td>VWR&#160;</td>
			<td style="width:119px;">BDH3028-2.5LG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodoacetamide&#160;&#160;</td>
			<td style="width:71px;">Sigma&#160;</td>
			<td style="width:119px;">I1149-5G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide (NaOH)</td>
			<td>Fisher</td>
			<td>S318-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin, sequencing grade, modified</td>
			<td>Promega</td>
			<td>V5113</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trifluoroacetic acid (TFA), LC-MS grade</td>
			<td>Fisher</td>
			<td>A116-50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td>Sigma</td>
			<td>U0631-500g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water, LC-MS ULTRA Chromasolv</td>
			<td>Fluka</td>
			<td>14263</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Custom synthesised peptides desalted 1-4mg</td>
			<td style="width:71px;">Genscript</td>
			<td style="width:119px;">custom</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">heavy labelled amino acid [C13 N15] custom peptides</td>
			<td style="width:71px;">Genscript</td>
			<td style="width:119px;">custom</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">ASB 14</td>
			<td style="width:71px;">Merck Millipore</td>
			<td style="width:119px;">182750-25gm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Thiourea</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T7875-500G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Tris base</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T6066</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">VanGuard precolumn</td>
			<td style="width:71px;">Waters</td>
			<td style="width:119px;">186007125</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Cortecs UPLC C18+ 1.6um&#160; 2.1 x50mm column</td>
			<td style="width:71px;">Waters</td>
			<td style="width:119px;">186007114</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Yeast Enolase&#160;</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">E6126</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">300ul clear screw top glass vials&#160;&#160;</td>
			<td style="width:71px;">Fisher scientific</td>
			<td style="width:119px;">03-FISV</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Y slit screw caps&#160;</td>
			<td style="width:71px;">Fisher scientific</td>
			<td style="width:119px;">9SCK-(B)-ST1X</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Freeze dryer</td>
			<td style="width:71px;">Edwards Mudulyo&#160;</td>
			<td style="width:119px;">Mudulyo system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Concentrator/Speed vaccum</td>
			<td style="width:71px;">Eppendof&#160;</td>
			<td style="width:119px;">concentrator&#160; plus 5301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Xevo -TQ-S mass spectrometer</td>
			<td style="width:71px;">Waters</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Acquity UPLC system</td>
			<td style="width:71px;">Waters</td>
			<td style="width:119px;"></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...

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

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

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

老鼠的眼睛剜除术的远程高通量的表型

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

科研

医学

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

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 ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

高通量顺序酶联免疫吸附试验的急性移植物抗宿主病的生物标志物的验证

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

量化衰老癌症细胞的敏感方法

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

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 ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

对专为递送治疗因素的神经保护策略成人干细胞高通量表征

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.

载脂蛋白 E 缺乏小鼠盐皮质激素受体的活化诱导动脉粥样硬化斑块的研究

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.