This work was performed on behalf of the International Federation of Clinical Chemistry Working Group on CSF Proteins, which has the following composition: Kaj Blennow (Chair) and Henrik Zetterberg, Gothenburg University, Sweden; Les Shaw and Magdalena Korecka, University of Philadelphia, PA, USA; Ingrid Zegers, Institute for Reference Materials and Measurements, Geel, Belgium; Piotr Lewczuk, Universit&#228;tsklinikum Erlangen, Germany; and Rand Jenkins, PPD Bioanalytical Laboratory, Richmond, VA, USA; Randall Bateman, Washington University, MO, USA; and H Vanderstichele, Innogenetics NV, Ghent, Belgium. This work was also part of the Global Consortium for Biomarker Standardization CSF Reference Methods Working Group, which is led by Maria C. Carrillo, Ph.D., Senior Director, Medical &amp; Scientific Relations, Alzheimer's Association. The study was supported by The Swedish Research Council (grants #14002, #521-2011-4709, and #2013-2546); the Knut and Alice Wallenberg Foundation; Demensf&#246;rbundet; and Emil and Wera Cornell's, Aina Wallstr&#246;m and Mary-Ann Sj&#246;blom's, Gun and Bertil Stohne's, Magnus Bergvall's, and Gamla Tj&#228;narinnor's Foundations.

注:此协议需要至少50微升的等分试样，对每个Aβ肽50微克/ mL的浓度，作为原料。 Aβ的肽应溶于20％乙腈（ACN）和4％浓氨溶液在去离子水（v / v）中并储存在80℃。 注意:请参阅表1的安全信息。 1.溶液的制备<ol><li>通过稀释20毫升乙腈和4毫升浓氨水（〜25％）在去离子水中制备100毫升20％的ACN和4％的在去离子水（v / v）的浓氨溶液。调节最终体积至100毫升去离子水。让每天新鲜。 </li><li>通过26.08克胍 - 盐酸盐溶解在去离子水至50毫升的最终体积制备50毫升，5M胍盐酸盐。在20°C储存，使新鲜的月刊。 </li><li>制备200毫升的4％磷酸在去离子水（v /ⅴ）通过稀释9.4毫升去离子水中的浓磷酸（〜85％）的。调节最终体积至200毫升去离子水。存放在冰箱，并每周新鲜。 </li><li>通过稀释37.5毫升乙腈和5mL浓氨水（〜25％）在去离子水中制备50毫升75％的ACN和10％的浓氨水溶液（体积/体积）在去离子水中。调节最终体积至50mL去离子水。让每天新鲜。 </li><li>解冻至少2.5 mL人CSF为校准器，从常规临床分析去确定剩余样品。 </li><li>制备含有150mM的Na，3.0毫钾，1.4毫米的Ca，0.8毫镁，1.0mM的磷，和155毫氯在去离子水中人工CSF和添加的牛血清白蛋白4毫克/毫升的最终浓度。只有1毫升每分析需要的，但制备大体积，等分它，并存储以供将来使用。 </li></ol> 2.校准器的研制<ol><li>准备的4微克0.5毫升/ 1毫升5NAβ1-42肽通过在0.5毫升微量离心管中加入40微升50微克/毫升15NAβ142至0.46毫升20％的ACN和4％浓氨水的。混合在涡旋混合器上于1分钟。 </li><li>通过在2毫升的离心管中加入50微升4微克/毫升15NAβ142至1.95毫升20％的ACN和4％浓氨水的制备2毫升100纳克/毫升15NAβ1-42肽。混合在涡旋混合器上于1分钟。 </li><li>由表2中所示的各溶液的体积混合准备六节校准溶液（AF）。使用0.5，1.5，和2mL微量离心管。混合在涡旋混合器上于1分钟。 </li><li>通过将相应的校准溶液，并根据表3混合在涡旋混合器上于1分钟人CSF制备于0.5mL微离心管中的最后的校准（一式两份）。 </li></ol> 3.内部标准的研制<ol><li>通过在2毫升的离心管中加入32微升50微克/毫升13CAβ142以1.968毫升20％ACN和4％浓氨水的制备2毫升0.8微克/毫升13CAβ1-42肽。混合在涡旋混合器上于1分钟。 </li><li>通过在5毫升的离心管中加入0.1毫升0.8微克/毫升至4.9毫升20％的ACN和4％浓氨水的制备5mL的16毫微克/毫升13CAβ1-42肽。混合在涡旋混合器上于1分钟。 </li></ol> 4.响应系数样品的制备注意:响应因子（RF）判定被执行以确定用于校准（15NAβ1-42）标记的肽的浓度。这就要求本机的Aβ1-42肽的浓度已经使用氨基酸分析（AAA）确定。因此，体积和天然的Aβ1-42肽的等分试样的浓度需要履行的AAA的要求。 <ol><li>通过以0.5毫升微量离心管中加入40微升50微克/ mL的原生的Aβ1-42至0.46毫升20％的ACN和4％浓氨水的制备0.5毫升4微克/毫升天然（未标记） 的Aβ1-42的。混合在涡旋混合器上于1分钟。 </li><li>通过加入4微克20微升/毫升天然的Aβ1-42和20微升为4μg/ mL的15NAβ1-42制备天然的和15NAβ1-42的2毫升40纳克/毫升组合，以1.96毫升的20％ ACN和4％在一个2毫升的离心管中浓氨。混合在涡旋混合器上于1分钟。 </li><li>添加40毫微克/毫升混合20微升至0.38毫升的人工脑脊液在0.5毫升离心管中。准备重复并在涡旋混合器上混合1分钟。 </li></ol> 5.样品制备注:融化样品在上辊室温进行测定。 <ol><li>添加每个校准器，响应因子，和未知样品（包括质量控制[QC]样品，如果使用的话），以1毫升的蛋白质的96深孔板的0.18毫升，根据图1（假设全板使用）。确保添加样品中，或接近，在孔的底部。 </li><li>加入内标20微升到每口井（ 即校准器，响应因子，QCS系统和未知）;关键的是要释放井靠近样品的表面侧的压降而不浸没枪头。 </li><li> 0.2毫升，5M胍盐酸添加到每个孔中。 </li><li>放置在微板振荡器上样品板并以1100rpm进行45分钟混合样品。最佳频率可能会因仪器不同。设定混频器的频率和振幅，以使溶液充分混合，没有内部标准或脑脊液滴留未混合在孔的一侧。 </li><li>加0.2毫升的4％磷酸到每个孔中。轻轻振荡混匀。 </li></ol> 6.固相萃取注:在所有的洗涤，装载和洗脱步骤，应用可能的最低的真空加入溶液后，并根据需要装载或洗脱溶液逐渐增加。禁用每个加载和洗脱步骤之间的真空。 <ol><li>置于废物贮存池托盘混合模式下，在萃取板歧管室的阳离子交换，96孔固相萃取（SPE）板。 </li><li>通过加入0.2毫升甲醇中以每孔调节在SPE吸附剂。 </li><li>通过加入0.2毫升的4％磷酸至每个孔平衡吸附剂。 </li><li>从深孔板转移所有样品（每孔约0.62 mL）添加到SPE板。样品从深孔板SPE板传送时使用8通道移液器;它不是传输所有样品的整个或相等体积至关重要的，因为样品中含有的实习生人的标准，将为变化进行补偿。 </li><li>洗吸附剂样品已通过加入0.2毫升的4％磷酸向每个孔穿过后。 </li><li>洗涤溶剂从吸附剂洗脱后，更换一个收集盘或管水库托盘。 </li><li>通过两次加入75％ACN / 10％浓氨水加入50μL洗脱从吸附剂样品，并注意此解决方案要求一个非常低的真空穿过吸附剂。记住要禁用每个另外的真空。 <ol><li>可选:密封收集板或管，并在-80℃冷冻。继续步骤6.8.2之前从收集板或管密封。 </li><li>干燥使用真空离心（不施加热量）洗脱液;这可以从一个到几个小时，取决于真空离心机。 </li><li>密封该容器，并在-80℃冷冻。 </li></ol></li></ol> 7.液体色谱造影<ol><li> 制备流动相A（5％ACN和0.3％浓氨水在去离子水[体积/体积]），B（4％去离子水和0.1％浓氨水在ACN [体积/体积]），并且针洗涤（50％ACN和4％浓氨水在去离子水[体积/体积]）。 <ol><li>为500毫升流动相A，加入25毫升乙腈和1.5毫升浓氨水，以去离子水。调节最终体积至500毫升去离子水。 </li><li>为500毫升流动相B，加500微升浓氨水和25毫升去离子水于乙腈。调节最终体积至500mL乙腈。 </li><li>通过加入120mL的乙腈和10mL浓氨水去离子水的制备250毫升洗针的。调节最终体积至250毫升去离子水。 </li><li>把流动相A，打开B和针头清洗瓶子在超声浴20分钟，LC系统使用前</li></ol></li><li>溶解有20 25微升％ACN和4％concentra各样品泰德氨水溶液，将其放在20分钟的振动筛。离心下来的样品，并放置在自动进样器（保持在7℃）。 </li><li> 注入上保持在50℃的1×250毫米2的聚苯乙烯-二乙烯基苯（反相）整体柱的样品20微升。 <ol><li>使用在表4中为0.3毫升/分钟的流速所示的LC梯度。转移的前两个和最后五分钟使用一个转向阀，以减少质谱仪的污染浪费（柱后）。 </li></ol></li></ol> 8.质谱分析注意:这些参数被用于配备aheated电喷雾源的四极的Orbitrap杂交质谱仪。 <ol><li>根据表5中设置用于离子源的参数。 </li><li>设置MS仪器来隔离本地Aβ1-42的4 +充电状态（按质量1,129.48电荷比[M / Z]），15NAβ1-42（1,143.00 M / Z），和13CAβ1-42（1,179.50 M / Z）中的四极质量分析器，用2.5 M / Z的隔离宽度。 </li><li>分段在碰撞单元中的分离的肽与17.0归一化碰撞能量（NCE）。这可能需要被调谐每个仪器，即使是同一类型的（尤其是如果使用其它类型的仪器，诸如三重四极杆质谱）。 </li><li>记录片段光谱和17.500的分辨率，为2×10 5个电荷的自动增益控制目标和250毫秒的最大喷射时间。 </li></ol> 9.数据处理<ol><li>在表6中使用的产品的离子的总和（具有±250毫质量单位[MMU]的质量公差）来计算的色谱区域为每个肽。需要注意的是离子种类和数量只显示了本机Aβ <suB&gt; 1-42产物离子，因为它们是为15NAβ1-42和13CAβ1-42相同。 </li><li>由15NAβ1-42的曲线（色谱峰）与天然Aβ1-42的曲线下的面积下，将该区域确定两个响应因子样本的平均响应因子。 </li><li>调整通过在步骤9.2计算出的响应因子乘以它用于校准的15NAβ1-42的浓度。 </li><li>通过从两组对浓度校准的标绘的15面积比NAβ1-42〜13CAβ1-42构建校准曲线。 </li><li>计算使用线性回归校正曲线的斜率和截距。 </li><li>计算机的Aβ1-42的面积比的内标（13CAβ1-42）为未已知样品。 </li><li>推断使用在步骤9.5中得到的斜率和截距由校准曲线未知样品的浓度。 </li></ol>

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JP and EP reports no disclosures. HZ has served on the advisory boards of Roche Diagnostics, Eli Lilly, and Pharmasum Therapeutics. KB has served as a consultant or on the advisory boards for Fujirebio Europe, IBL International, and Roche Diagnostics. HZ and KB are co-founders of Brain Biomarker Solutions in Gothenburg AB, a GU Venture-based platform company at the University of Gothenburg.

对于所描述的方法，而不是使用一个代理矩阵，我们使用了替代的分析物的方法13，14，15，16，它使校正在人CSF。代孕的方式分析涉及到两个不同的同位素标记的标准。酮（15NAβ142）被用来产生于人类CSF中的校准曲线，而另一个（13CAβ142）用作内标。然后未知的内源性...

阿尔茨海默氏病（AD）是老年痴呆症的最常见的形式，影响了全球100约35万人。疾病广泛认为在躺在床上的AD发病的芯的神经病学特征是tau蛋白的蛋白2的细胞内神经原纤维缠结和细胞外斑块由聚集的淀粉样-β（Aβ）肽3。本着这一精神，体内斑块病理的生物标志物评估最近已包含在公元4研究的诊断标准。对于Aβ1-42的CSF测量，几个免疫测定可以使用，在许多临床实验室5被使用。 的Aβ1-42的CSF中的浓度是在AD患者比在认知正常老年人下大约50％，这反映了肽在斑中的BR的沉积AIN 6,7。 这些生物标志物重点分析使用免疫（ 即，基于抗体的技术），但这些试验可以由基体效应8的影响。使用不同技术平台的免疫测定和缺乏测定标准化9，10，使引入全球截止浓度难以11,12。一种分析验证RMP将允许不同的检测平台统一校准，最好导致跨平台的分析比较好和更好的控制有助于整体测量变异的因素。 使用开发的LC-MS / MS方法的Aβ1-42的绝对定量克服了与基于抗体的TECHN有关的问题iques。该方法被列为联合委员会溯源实验室医学（JCTLM数据库标识号C11RMP9），一个RMP将被用来确定一个标准物质（CRM）Aβ1-42的绝对浓度协调CSFAβ1跨越技术和分析平台-42测量。所描述的工作流程应该是相关的用于医学的其他领域内的肽和蛋白质候选参考方法的发展。

<table><tbody><tr><td>Eppendorf Protein LoBind microcentrifuge tubes, 0.5 mL</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Eppendorf Protein LoBind microcentrifuge tubes, 1.5 mL</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Eppendorf Protein LoBind microcentrifuge tubes, 2 mL</td><td>Eppendorf</td><td>022431102</td><td></td></tr><tr><td>Eppendorf Protein LoBind microcentrifuge tubes, 5 mL</td><td>Eppendorf</td><td>0030 108.310</td><td></td></tr><tr><td>Eppendorf Protein LoBind Deepwell Plates 96</td><td>Eppendorf</td><td>951032905</td><td></td></tr><tr><td>Micronic 0.75 mL polypropylene tubes, V-bottom</td><td>Micronic</td><td>MPW32071BC3</td><td>Tubes used for collecting SPE eluate, but other collection devices can be used such as 96-well plates.</td></tr><tr><td>Micronic Split TPE Capcluster for 96 ind. tubes</td><td>Micronic</td><td>MP53026</td><td>Caps for Micronic tubes.</td></tr><tr><td>Micronic Loborack-96 white w high cover bar coded</td><td>Micronic</td><td>MPW51015BC3</td><td>Holder for Micronic tubes.</td></tr><tr><td>Biohit Optifit tips 1.2 mL</td><td>Sartorius</td><td>791210</td><td>Used to reach all the way to the bottom of the LoBind deepwell plates (96-well). Other narrow tips might work as well.</td></tr><tr><td>Waters Oasis MCX 96-well &amp;micro;Elution Plate</td><td>Waters</td><td>186001830BA&amp;nbsp;</td><td></td></tr><tr><td>Waters Plate manifold reservoir tray</td><td>Waters</td><td>WAT058942</td><td></td></tr><tr><td>Waters Extraction Plate Manifold for Oasis 96-Well Plates</td><td>Waters</td><td>186001831</td><td></td></tr><tr><td>Ammonium hydroxide solution, puriss. p.a., reag. ISO, reag. Ph. Eur., ~25% NH3 basis</td><td>Sigma-Aldrich</td><td>30501-1L-D</td><td></td></tr><tr><td>Acetonitrile, Far UV HPLC Gradient grade, 2.5 L</td><td>Fisher Scientific</td><td>A/0627/17X</td><td></td></tr><tr><td>ortho-Phosphoric acid 85%, ACS,ISO,Reag. Ph Eur</td><td>Merck Millipore</td><td>1005731000</td><td></td></tr><tr><td>Guanidine Hydrochloride, 500 g</td><td>Thermo Scientific</td><td>24110</td><td></td></tr><tr><td>Thermo Scientific Q-exactive Hybrid Quadrupole Orbitrap Mass Spectrometer</td><td>Thermo Scientific</td><td>IQLAAEGAAPFALGMAZR</td><td>With bundled Dionex Ultimate 3000 LC &amp;amp; autosampler.</td></tr><tr><td>Dionex ProSwift RP-4H Monolith Column (1.0 x 250mm)</td><td>Dionex</td><td>066640</td><td></td></tr><tr><td>Bovine Serum Albumin, lyophilized powder, suitable for (for molecular biology), Non-acetylated</td><td>Sigma-Aldrich</td><td>B6917</td><td></td></tr><tr><td>Beta-Amyloid (1-42), Ultra Pure, TFA</td><td>rPeptide</td><td>A-1002-2</td><td></td></tr><tr><td>15N Beta-Amyloid (1-42), Uniformly labeled</td><td>rPeptide</td><td>A-1102-2</td><td></td></tr><tr><td>13C Beta-Amyloid (1-42), Uniformly labeled</td><td>rPeptide</td><td>A-1106-2</td><td></td></tr><tr><td>Microplate shakers, TiMix 2</td><td>Edmund B&amp;uuml;hler</td><td>6110 000</td><td></td></tr></tbody></table>

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.

图1中的板设置用于样品的完整板。如果较少的未知样本进行分析，所述第二校准器，RF和QC套应置于未知样品的前半部分后。 如在图2中看到的那样，校准接近回归线，具有低标准偏差。这种方法具有150皮克/毫升和4000皮克/毫升的量化的上层的定量的较低水平。当然校准的剩余标准偏差应尽可能低。如...

Watch this Scientific Journal Video about Absolute Quantification of AÎ²1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure at JoVE.com

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

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 ...

absolute-quantification-a1-42-csf-using-mass-spectrometric-reference

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

7.5K 次观看.  The Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University Hospital. A reference measurement procedure for the absolute quantification of Aβ1-42 in human CSF based on solid-phase extraction and liquid chromatography tandem mass spectrometry is described.

视频: Absolute Quantification of Aβ1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

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

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

一个定制HPLC纯化协议，产量高纯度淀粉样蛋白β42和贝塔淀粉样蛋白肽40，有能力寡聚物形成的

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

生物化学

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

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

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

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 ...

医学

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 ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

一种改进的麻醉小鼠脑脊液采集方法

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

矩阵辅助激光去离子成像质谱法显示人脑中的淀粉样β矿床

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

小鼠体内合成蛋白的定量测量

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral A&#946; plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the A&#946; load in brain tissues derived from AD transgenic mice. Though the methods for A&#946; detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on A&#946; load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the A&#946; load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of A&#946; load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on A&#946;-positive area quantification, and show a strong correlation between the A&#946;-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread A&#946; deposition.

The present protocol describes and compares the procedure to perform a full-region or sub-region of interest analysis of sagittal mouse brain sections to quantify amyloid-beta load in the APP/PS1 transgenic mouse model of Alzheimer's disease.

小鼠脑切片上淀粉样蛋白-β负荷的全区域与亚区域定量

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of ...

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 ...

Aβ的绝对定量

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