Name: hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins
Uploaded: 2014-08-07T20:45:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

迈克尔·亨利，都柏林城市大学，请协助与图5所示的数据收集和分析，这项工作得到了部分支持（1）乔治·梅森大学，（2）意大利IstitutoSuperiore二SANITA"在意大利/美国框架健康的乔治·梅森大学的美国能源部和人类服务部，以及公共卫生意大利外交部，（3）美国国立卫生研究院，IMAT计划资助1R21CA137706-01和1R33CA173359-01对上湖人，以及（4）谷神星纳米科学公司之间的合作协议。

人血浆和尿液是从健康志愿捐献者采集，并以书面的知情同意书，下面的乔治·梅森大学机构审查委员会批准的协议。捐助者平均分配白人男性和女性之间的25和42样品进行单独分析岁之间，并没有合并。 血清或血浆样品1纳米工艺在等离子体电势低丰度生物标记物被捕获，在溶液中，用凝胶粒子。将粒子加入到等离子体中，温育，通过离心分离，洗涤，和所捕获的蛋白被洗脱。将洗脱的蛋白质根据用于下游质谱测序和鉴定的氮气流中干燥。 <ol><li>在离心管2与50毫米TrisHCl pH值为7（500微升血+500微升TrisHCl）的稀释:500微升血清1。 </li><li>加入500μl聚（异丙基丙烯酰胺/ AAC）核心nanop的文章。孵育15分钟，在室温。 </li><li>在装有固定角转子的离心旋转的样品在16,100 XG，25℃下进行10分钟。拆下并丢弃上清液。 </li><li>加入500μl加入硫氰酸钠（25毫摩尔）的粒料。通过剧烈上下吹打多次重悬的纳米颗粒。 </li><li>在具有固定角度转子离心机旋转样品在16,100 XG，25℃下进行10分钟。拆下并丢弃上清液。 </li><li>加入500微升的MilliQ水的纳米颗粒沉淀。通过剧烈上下吹打多次重悬的纳米颗粒。 </li><li>在具有固定角度转子离心机旋转样品在16,100 XG，25℃下进行10分钟。拆下并丢弃上清液。 </li><li>准备新鲜的洗脱缓冲液:加入700μL乙腈300μL氢氧化铵在一个干净的试管中。注意:氢氧化铵具有腐蚀性。使用适当的通风。注意:洗脱缓冲液必须准备ARED即将使用前。请勿将洗脱缓冲液。 </li><li>加入300微升的洗脱缓冲液中的纳米颗粒。通过剧烈上下吹打多次重悬的纳米颗粒。孵育15分钟，在室温。 </li><li>在具有固定角度转子离心机旋转样品在16,100 XG，25℃下进行10分钟。取出并保存洗脱液在干净，标记的离心管。 </li><li>重复步骤1.9 - 1.10。二者结合起来洗脱液为一体的离心管。 </li><li>干燥下，在氮气蒸发器歧管的氮气流的洗脱液在42.1℃，用空气流设置为8（ 图2F）。 </li><li>储存在室温下干燥洗脱液为O / N个存储，或在-20℃下长期贮存，前质谱法，免疫印迹，或酶联免疫吸附试验（可选）。 </li></ol>尿液样本2纳米工艺正常尿液中含有低于30毫克/分升的蛋白质和比1 +血少。然而，很多疾病/状况可能改变尿蛋白和血液的正常水平。为了帮助确定纳米颗粒的最佳量添加到尿样，尿分析之前进行纳米粒子的收获进行。尿液生物标志物可以存在于非常低的浓度，这可能需要优化的纳米颗粒的比对尿量。此过程说明了下游Western blot分析尿液样本的纳米粒子收获。 <ol><li>收集尿液样本中的清洁，干燥的塑料杯。 à22毫升最小的体积是必需的。商店尿液标本在-80°C，直到准备分析。 </li><li>解冻的冷冻尿液在室温或4°CO / N。在旋涡混合器简要组合。倾至少22毫升尿入50毫升的锥形底聚丙烯管中。 </li><li>旋尿中有水平转子离心机在3700 XG 15分钟。倒出尿液，而不会干扰颗粒，放入干净的50ml锥形底polypropylene管。弃沉淀。 </li><li>使用多分析物"尿液试纸"的试剂条进行尿检。注意事项:避免阳光直射，湿度17，18的试纸存放在密闭的容器了。 <ol><li>莱试剂条，面朝上，放在干净，干燥的纸巾。 </li><li>使用一次性吸管吸1毫升步骤2.3准备的尿液。 </li><li> 2分钟设置一个计时器，但不启动它。快速分配1 - 在试剂条的每个测试点2滴尿。立即启动定时器。注意:不要直接在尿液的容器浸入试剂条。在试剂条上的染​​料/化学指标可以潜在浸出试剂条的进入尿液的容器中。 </li><li>在使用的试剂条容器所指示的时间，由单独的试剂条的颜色进行比较，对相应的颜色编码的籼稻记录定性结果的试剂条上的各种分析物器试剂容器上。典型的正常尿的结果是:（正常的或负的）的血液，蛋白质，白细胞，亚硝酸盐，葡萄糖，酮，胆红素和尿胆素原;尿pH值（5.5 - 7.0）;比重（1.001 - 1.020）。注意:高蛋白质水平在尿液样本可以与感兴趣的蛋白质竞争结合于所述纳米颗粒的站点。为了最大限度地提高蛋白质的收获与纳米颗粒，该纳米颗粒体积/尿体积比率可以调整为从高丰度蛋白质或者限制竞争，或者可以被优化到收获中存在的浓度非常低的蛋白质。如果尿蛋白值是1 +或更高，在下面的步骤中添加2.5纳米的2倍量。如果感兴趣的分析物是非常低丰度蛋白质，它可能是必要的，以增加纳米颗粒的量最多至2ml（图3）。 </li></ol></li><li>将20毫升的尿液澄清步骤2.3到一个干净的50ml锥形底聚丙烯管。别扰乱任何碎片/丸粒可能在尿液管的底部。加入200μl纳米颗粒的20毫升尿样。在旋涡混合器简要组合。注意:如果尿蛋白值是1 +或更高，加入400μl的纳米颗粒到20ml的尿液。 </li><li> 30分钟，在室温下孵育尿/纳米颗粒的混合物，无摆动/混合。 </li><li>旋尿/纳米颗粒悬浮液在配备水平转子在3700离心10 min的离心机。注意:如果粒料是不可见的离心后，旋转试样进行一个额外的2 - 7分钟。 </li><li>拆下并丢弃上清液。加入500μl的MilliQ水的纳米颗粒沉淀。 </li><li>通过剧烈上下吹打多次重悬的纳米颗粒。纳米粒子溶液转移到一个干净的1.5 ml离心管。 </li><li>纺纳米颗粒在装备有固定角度转子以16,100 xg离心10分钟，离心。注意:如果小球是不可见的甲级降脂离心，旋转试样进行一个额外的2 - 7分钟。 </li><li>拆下并丢弃上清液。 </li><li>重复步骤2.8和2.11（两次），用200μl18的MilliQ水冲洗该纳米颗粒。 </li><li>准备新鲜的洗脱缓冲液:加入970μL乙腈至30微升氢氧化铵在一个干净的试管中。注意:氢氧化铵具有腐蚀性。使用适当的通风。注意:洗脱缓冲液必须临用前制备。请勿将洗脱缓冲液。 </li><li>加入20微升的洗脱缓冲液的纳米颗粒沉淀。通过剧烈上下吹打多次重悬的纳米颗粒。孵育15分钟，在室温。 </li><li>纺纳米颗粒样品在16100×g离心10分钟。取出并保存在上清干净，1.5 ml离心管。不破坏纳米颗粒。丢弃在生物危害的垃圾沉淀。 </li><li>将样品在通风橱机架。打开瓶盖和公司ubate在室温30分钟。或者，将下在40℃的氮气流中的样品直至干燥（1 - 2小时）。 </li><li>加入15微升的Tris-甘氨酸SDS样品缓冲液（2×）的样品。热火100 ℃，在5分钟的盖子，直至留在管内的体积的干热块是不超过20微升。注意:不要用沸水浴中。从水浴中的湿度会防止缓冲器的蒸发。 </li><li>广场上的管帽。从加热块中取出试管。可将样品保存在-80℃下或立即用于下游免疫印迹分析。 </li></ol>

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F., Refson, J., Demoliou-Mason, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-derived+growth+factor+(PDGF):+actions+and+mechanisms+in+vascular+smooth+muscle.">Platelet-derived growth factor (PDGF): actions and mechanisms in vascular smooth muscle.</a> Gen Pharmacol. 27 (7), 1079-1089 (1996).</li><li>Izycki, T., 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=Serum+levels+of+IGF-I+and+IGF-II+in+patients+with+lung+cancer+during+chemotherapy.">Serum levels of IGF-I and IGF-II in patients with lung cancer during chemotherapy.</a> Exp Oncol. 26 (4), 316-319 (2004).</li><li>Jalosinski, M., Karolczak, K., Mazurek, A., Glabinski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+methylprednisolone+and+mitoxantrone+on+CCL5-induced+migration+of+lymphocytes+in+multiple+sclerosis.">The effects of methylprednisolone and mitoxantrone on CCL5-induced migration of lymphocytes in multiple sclerosis.</a> Acta Neurol Scand. 118 (2), 120-125 (2008).</li><li>Kitazume, S., 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=Molecular+insights+into+beta-galactoside+alpha2,6-sialyltransferase+secretion+in+vivo.">Molecular insights into beta-galactoside alpha2,6-sialyltransferase secretion in vivo.</a> Glycobiology. 19 (5), 479-487 (2009).</li><li>Saito, T., 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=Increase+in+hepatic+NKT+cells+in+leukocyte+cell-derived+chemotaxin+2-deficient+mice+contributes+to+severe+concanavalin+A-induced+hepatitis.">Increase in hepatic NKT cells in leukocyte cell-derived chemotaxin 2-deficient mice contributes to severe concanavalin A-induced hepatitis.</a> J Immunol. 173 (1), 579-585 (2004).</li><li>Struyf, S., 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=PARC/CCL18+is+a+plasma+CC+chemokine+with+increased+levels+in+childhood+acute+lymphoblastic+leukemia.">PARC/CCL18 is a plasma CC chemokine with increased levels in childhood acute lymphoblastic leukemia.</a> Am J Pathol. 163 (5), 2065-2075 (2003).</li><li>Michael, I. P., 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=Human+tissue+kallikrein+5+is+a+member+of+a+proteolytic+cascade+pathway+involved+in+seminal+clot+liquefaction+and+potentially+in+prostate+cancer+progression.">Human tissue kallikrein 5 is a member of a proteolytic cascade pathway involved in seminal clot liquefaction and potentially in prostate cancer progression.</a> J Biol Chem. 281 (18), 12743-12750 (2006).</li></ol>

Benjamin Espina is an employee of Ceres Nanosciences, Inc. that produces reagents used in this Article. Lance Liotta, Alessandra Luchini and Virginia Espina hold patents (US patent 7,935,518 and/or 8,497,137) on the nanoparticle technology used in this Article. As university employees they are entitled to receive royalty from these patents per university policy. Lance Liotta and Alessandra Luchini are shareholders in Ceres Nanosciences and serve on the Scientific Advisory Board.

临床意义 血清或血浆样品被认为含有低丰度循环的蛋白质和多肽可提供的信息有关的生物体作为一个整体的国家的丰富来源。尽管血清蛋白质组学的承诺，有三个基本的和严重的生理障碍，阻碍生物标志物的发现和翻译临床获益10，11，16，25。 一，重要诊断标志物可能存在于非常低丰度（浓度）在血液中。早期病变组织，例如预转?...

尽管完成了人类基因组测序，显著进展尚未作出确定的生物标志物预测的早期阶段的疾病，或与治疗结果，或预测1相关。其中一个原因缺乏进展是，很多潜在的生物标志物显著以低于传统质谱和其他生物标志物发现平台的检测极限浓度存在。质谱（MS）和多反应监测（MRM）具有典型的检测灵敏度大于50纳克/毫升，而在50 pg / ml和10毫微克/毫升在临床实验室下降了免疫测定的范围广大的分析物的。这意味着许多生物标志物，特别是在疾病的早期阶段无法通过常规的MS和MRM 2被检测到。除了高丰度蛋白质，如白蛋白和免疫球蛋白的复合物的生物流体的存在往往掩盖由十亿倍货存ESS的低丰度，低分子量蛋白质和肽3，4。为此几个样品的准备步骤之前所需要质谱测序和鉴定。一个这样的预备步骤中采用的高丰度蛋白质的与市售的耗尽列5-8的耗尽。不幸的是这一步骤导致候选生物标志物的产率的降低，因为它们通常是非共价地与该被除去的载体蛋白相关联。另一个挑战是候选生物标志物前体的稳定性表示，一旦样品的采集。蛋白质是发生降解内源性或外源性蛋白酶9。水凝胶的纳米颗粒可以通过扩增推定的生物标志物的浓度，以测定的范围内的电平超越这些关键的挑战，同时保护蛋白免受降解10-13。 要注意个很重要在LMW在血液中的蛋白质是小完整蛋白质以及大蛋白的片段的混合物。组织来源的蛋白质比60 kDa的较大的过大而不能穿过血管基底膜被动地进入血流，但它们可以在血液中作为肽或蛋白质片段14来表示。我们的目标是测量新颖循环生物标志物，可以是候选疾病的早期发现，患者分层为治疗，并监测对治疗的反应。在创建了纳米颗粒选择性地排除高丰度的免疫球蛋白和白蛋白，同时捕获较小的蛋白质和肽，并根据初始体积上集中起来，以100倍。 我们的团队确定了一套小的有机染料，可成功地用作高亲和力的分子饵料蛋白质和多肽。蛋白质 - 染料的结合被认为是由于疏水性和静电相互作用的结合第对染料的芳环交织经由蛋白质上的表面11的疏水口袋的蛋白质。 的毒饵，这取决于它们的化学性质，表现出分析物的选择的类的特定亲和力。诱饵与载体蛋白竞争，如白蛋白，用于蛋白质或肽。低分子量蛋白/肽被截留在颗粒中。高分子量的蛋白质，如白蛋白和球蛋白是从进入的，因为筛分能力的粒子可防止因水凝胶11（图1）的限制孔。 水凝胶粒子是通过由过硫酸铵11开始沉淀聚合合成。异丙基丙烯酰胺（NIPAM），丙烯酸（AAC）和烯丙基胺（AA）和交联剂N，N&#39;-亚甲基双（BIS）的共聚单体被允许在70℃下反应6小时，在稀释条件11,13。聚的高蛋白结合亲和性（N-异丙基丙烯酰胺-共聚-丙烯酸）（聚（NIPAM-CO-AAC）nanoparticlesis通过共价掺入含氨基的染料（ 即 ，sulfonatedanthraquinonetriazine染料）在纳米粒子，通过实现在这取决于染料的亲水/疏水特性的水性或有机溶剂中进行的酰胺化反应如图11所示，用一个anthraquinonetriazine染料的氯原子在纳米颗粒中的胺基团的13。亲核取代被用于创建聚含染料（NIPAM -共聚-丙烯胺）（AA）的纳米颗粒11，12。的两步聚合方法被用来创建含有乙烯基磺酸（VSA）11，13的外壳的水凝胶粒子。 水凝胶纳米粒子可用于各种生物流体，包括全血，血浆，血清，脑脊液，汗液，尿等。在一个步骤中，在溶液中，nanoparticles进行快速（几分钟内）封存和低分子量的分析物浓度10，11，13，15-18。蛋白质随后从纳米颗粒洗脱和检测用蛋白质印迹19-21，质谱法10，11，13，15，18，22，23，免疫/ ELISA 10，11，15，18，或反相蛋白质微阵列16， 24测定。纳米功能化化学饵料，并提出核或核 - 壳结构，捕捉和集中的基础上饵/壳的物理化学性质的蛋白质。因此，掺入纳米颗粒不同的染料将捕获的蛋白质的不同亚群具有不同的基础上，染料亲和效率，溶液的pH，并且存在/不存在竞争的高丰度蛋白13。此外，纳米颗粒的相对量，以将溶液的体积将影响从纳米颗粒中的蛋白质的产率。这些方面水凝胶的纳米粒子的收获是使用三种不同的纳米颗粒饵收割蛋白是由含有高含量的蛋白质的血浆样品显示出，并从尿样品通常不包含大量的蛋白质。在这个协议中，我们证明收获和浓缩使用聚（NIPAM-CO-的AAc），聚（NIPAM /染料），以及核 - 壳纳米颗粒的血浆样品的肿瘤坏死因子α（TNFα）（聚（NIPAM-CO-VSA）） 。聚（NIPAM /染料）的纳米颗粒被示为集中于加入人尿样品分枝杆菌抗原，以模仿结核分枝杆菌感染的个体。

<table><tbody><tr><td>Hydrogel nanoparticles</td><td>Ceres Nanoscience</td><td>CS003</td><td>NanoTrap ESP particles</td></tr><tr><td>18 M&amp;Omega;-cm water</td><td></td><td></td><td>Type 1 reagent grade water</td></tr><tr><td>Tris HCl, 50 mM pH 7.0</td><td>VWR</td><td>IC816116</td><td>50 mM, pH 7</td></tr><tr><td>Acetonitrile</td><td>BDH</td><td>BDH1103-4LP</td><td>Available from VWR</td></tr><tr><td>Ammonium Hydroxide NH&lt;sub&gt;4&lt;/sub&gt;OH</td><td>BDH</td><td>BDH3014</td><td>Available from VWR, assayed at 28-30% NH&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Sodium thiocyanate 25 mM</td><td>Acros Organics</td><td>419675000</td><td>For serum/plasma samples</td></tr><tr><td>Multi-analyte Urine Reagent Strips</td><td>Siemens</td><td>2161</td><td>For urine samples</td></tr><tr><td>Tris-Glycine SDS Sample Buffer (2X)</td><td>Life Technologies</td><td>LC2676</td><td>Use at RT to prevent SDS from precipitating</td></tr><tr><td>Dry bath incubator (100 &lt;sup&gt;o&lt;/sup&gt;C) with heating block</td><td>Barnstead</td><td>11-715-125DQ</td><td>Do not substitute a boiling water bath</td></tr><tr><td>Nitrogen evaporator manifold</td><td>Organomation Associates</td><td>Microvap118</td><td>For serum/plasma samples</td></tr><tr><td>Centrifuge, swing-out rotor</td><td>Sorvall</td><td>Legend series</td><td>50 ml tube capacity, rcf 3,700 x g</td></tr><tr><td>Centrifuge, fixed angle rotor</td><td>Eppendorf</td><td>5424</td><td>1.7 ml microcentrifuge capacity, rcf 16,000 x g</td></tr><tr><td>50 ml conical centrifuge&amp;nbsp;tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td>With screw caps for urine samples</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>Eppendorf</td><td>22363204</td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td>50-949-755</td><td></td></tr><tr><td>Timer</td><td>Fisher Scientific</td><td>S04782</td><td>Seconds/minutes</td></tr></tbody></table>

hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins

新型生物标志物的发现在提供更多的敏感性和特异性疾病检测至关重要的作用。不幸的是许多存在于生物流体中低丰度生物标记物不能容易地与质谱法或免疫测定法，因为它们存在于非常低的浓度，是不稳定的，并且往往是由高丰度蛋白质，如白蛋白或免疫球蛋白掩蔽检测。诱饵含聚（N-异丙基丙烯酰胺）（NIPAM）基于纳米颗粒是能够克服这些生理屏障。在一个步骤中，他们能够捕捉，集中和保持体液的生物标记物。低分子量的分析物进入所述纳米颗粒的核心，由不同的有机的化学染料，它作为高亲和力蛋白诱饵被捕获。该纳米颗粒可以由几个数量级集中感兴趣的蛋白质。这个浓度因子是足以增加蛋白质水平，使得蛋白质内的目前质谱仪，免疫印迹和免疫检测限。纳米颗粒可以一起孵育过多的生物体液和它们能够极大丰富的低分子量蛋白质和肽的浓度，同时排除白蛋白和其它高分子量的蛋白质。我们的数据表明，在特定分析物的浓度的10,000倍放大，可以实现，使质谱和免疫检测以前无法检测的生物标志物。

水凝胶纳米粒子大小和均匀度 聚（NIPAM-AAC）颗粒已产生极高的收益率和可重复性的和批次内。所述颗粒具有在RT非常好的胶体稳定性期间需要用于捕获，存储，和蛋白质的洗脱（至少48小时）的时间，以及纳米颗粒沉淀没有被观察到（ 图1）11。胶体稳定性可能是由该纳米颗粒快速蛋白质/肽的摄取很重要。 从血浆?...

Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

hydrogel-nanoparticle-harvesting-plasma-or-urine-for-detecting-low

Research

JoVE Journal

Bioengineering

13.8K 次观看.  George Mason University. Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

视频: Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

科研

生物工程

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

生物化学

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

一种改进沉淀法来隔离尿外来体

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

医学

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

纳米材料获得的蛋白质和代谢物科罗纳的毛细电电量谱方法

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

化学

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

基于靶向质谱的低丰度蛋白质蛋白分析中的微量采样

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

自动疏水层析蛋白质纯化用柱选择

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

生物学

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

用于细菌筛选、选择和分离的可光降解水凝胶接口

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Enrichment of Astrocyte-Derived Extracellular Vesicles from Human Plasma

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a vast range of functions by acting on and transferring their cargos to other cells in physiological and pathological conditions. Given their presence in biofluids, EVs represent an excellent resource for studying disease processes and can be considered a liquid biopsy for biomarker discovery. An attractive aspect of EV analysis is that they can be selected based on markers of their cell of origin, thus reflecting the environment of a specific tissue in their cargo. However, one of the major handicaps related to EV isolation methods is the lack of methodological consensuses and standardized protocols. Astrocytes are glial cells with essential roles in the brain. In neurodegenerative diseases, astrocyte reactivity may lead to altered EV cargo and aberrant cellular communication, facilitating/enhancing disease progression. Thus, analysis of astrocyte EVs may lead to the discovery of biomarkers and potential disease targets. This protocol describes a 2-step method of enrichment of astrocyte-derived EVs (ADEVs) from human plasma. First, EVs are enriched from defibrinated plasma via polymer-based precipitation. This is followed by enrichment of ADEVs through ACSA-1-based immunocapture with magnetic micro-beads, where resuspended EVs are loaded onto a column placed in a magnetic field. Magnetically labeled ACSA-1+ EVs are retained within the column, while other EVs flow through. Once the column is removed from the magnet, ADEVs are eluted and are ready for storage and analysis. To validate the enrichment of astrocyte markers, glial fibrillary acidic protein (GFAP), or other specific astrocytic markers of intracellular origin, can be measured in the eluate and compared with the flow-through. This protocol proposes an easy, time-efficient method to enrich ADEVs from plasma that can be used as a platform to examine astrocyte-relevant markers.

This protocol describes the enrichment of astrocyte-derived extracellular vesicles (ADEVs) from human plasma. It is based on the separation of EVs by polymer precipitation, followed by ACSA-1 based immunocapture of ADEVs. Analysis of ADEVs may offer clues to study changes in inflammatory pathways of living patients, non-invasively by liquid biopsy.

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a ...