Name: a high throughput, multiplexed and targeted proteomic csf assay to quantify neurodegenerative biomarkers and apolipoprotein e isoforms status
Uploaded: 2016-10-20T14:00:00
Description: 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

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<s...

<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>

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。

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		<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 ...

Research

JoVE Journal

Medicine

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老鼠的眼睛剜除术的远程高通量的表型

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

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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

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

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

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

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

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Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay ...