这项研究得到了来自 CSU (KMD)、ATCC 合同 #2016 0550-0002 (NIAID HHSN272201600013C 分包)、比尔和梅琳达盖茨基金会 (OPP1039688) (KMD/NKG) 的内部资金的支持。我们感谢美国国家科学基金会在科罗拉多州立大学 (拉吴) 暑期项目的研究经验, 以得到更多的支持。

这项工作在科罗拉多州立大学的机构审查委员会 (IRB) 初步审查后被确定为非人类专题研究, 因为所有的人类样本都是从 Bioreclamation IVT biorepository 中提取出来的样本, 并根据 IRB 批准的协议收集。
1. 通过造粒较大的囊泡和细胞碎片制备粗样品 (血浆或血清)
注意:安全考虑: 血浆、血清和其他生物液体是 biohazardous 材料, 必须特别小心处理, 同时佩戴基本的个人防护设备, 包括手套和实验室大衣。强烈建议生物样品在生物安全柜中处理;如果不可用, 建议使用眼睛保护。
<ol><li>平衡在4摄氏度冰箱 (从-20 或-80 °c 贮存) 中, 将管置于非震动条件下, 将血清或血浆样品放在一夜之间。</li>
	<li>整除250µL 的样品进入离心管, 使用1000µL 吸管。</li>
	<li>离心样品在 1.8万 x g 30 分钟在4°c。</li>
	<li>使用200µL 吸管, 移除200µL 的清除上清液, 并将其添加到新的离心管。在转移上清时避免任何颗粒物质。丢弃颗粒材料。</li>
	<li>使用制造商的协议, 定量测定样品中的蛋白质含量 (二辛可宁酸)。要进行化验, 稀释1:50 在1x 磷酸盐缓冲盐水 (PBS) 样品。以重复的方法测试每个示例。 注意:在整个协议中使用 1x PBS, 除非另有说明。平均, 200 µL 的澄清血清或血浆 (1.8万 x g) 将产生15毫克的总蛋白。其他样本类型, 蛋白质含量较低或较高, 可能需要额外的稀释。</li>
	<li>整除10毫克的样品进入一个新的离心管。使用步骤1.5 中获得的浓度来计算相应的样品体积。用200µL 吸管整除样品。 注意:建议将剩余的样品保存在摄氏-80 摄氏度。整除以每管10毫克的不同管子为例, 以避免今后使用多种冻融循环。</li>
</ol>2. 非 exosomal 血蛋白的消化
注意:消化步骤旨在减少与外来体共同纯化的非 exosomal 蛋白。如果下游分析需要保存外切表面蛋白, 应考虑到这一步骤有可能干扰这种分析。exosomal CD63 的检测是一种表面暴露的 tetraspanin 蛋白, 对此过程没有显著的负面影响。
<ol><li>在 PBS 中500µg/毫升的浓度下制备蛋白酶 K 溶液。在圆锥管中加入相应数量的蛋白酶 K。然后加入缓冲和漩涡在三十年代, 3x 在室温下。</li>
	<li>添加50µL 蛋白酶 K 溶液的10毫克样品整除, 并轻轻地混合吹打上和下使用200µL 吸管。</li>
	<li>在37摄氏度的水浴中孵化样品30分钟. 将管子放在浮子管架中, 避免在水浴中完全浸入管内, 以避免潜在的渗漏。</li>
	<li>将样品和浮动管架转移到60°c 水浴中10分钟, 使蛋白酶 K 失效。</li>
</ol>3. 离心超滤去除小蛋白和多肽
<ol><li>在使用前, 用 PBS 冲洗含有 100 kDa 分子量截止 (截留分子量) 过滤器的超滤装置。通过将500µL 的 PBS 添加到过滤器中, 使用1000µL 吸管。旋转设备在 3700 x g 5 分钟丢弃任何剩余的 retentate 和洗脱液。 注意:超滤装置的样品容积容量必须为0.5 毫升-4 毫升, 以确保最终减少的样品体积为50µL 是可以实现的。</li>
	<li>从步骤2.4 中抽取样本, 并通过添加 PBS 将卷增加到500µL。将样品注入预冲洗的超滤装置。 注意:平均来说, 从步骤2.4 的样本量是185µL (120 到150µL 的样品和50µL 的蛋白酶 K 溶液)。</li>
	<li>将超滤装置放置在固定角度的台式离心机上, 在 3700 x 克4摄氏度, 直到样品减少到50µL 的体积。 警告:当使用超滤装置时, 必须注意避免过滤膜在离心过程中完全干燥。</li>
	<li>将500µL 的 PBS 直接添加到滤膜中, 吸液器上下5次。离心机和步骤3.3。执行此洗涤步骤共3次。</li>
	<li>将最终的50µL retentate 转移到新的离心管中。</li>
	<li>用200µL PBS 冲洗滤膜, 吹打上下10次, 从步骤3.5 将洗涤液转到管。</li>
	<li>将滤芯置于新管的反位置。运行反向旋转恢复5分钟在 2000 x g 检索剩余的样品从膜。将示例与步骤3.5 中的示例一起进行池。</li>
</ol>4. 粒度排除色谱法纯化囊泡 (秒)
<ol><li>添加850µL 的截留分子量浆, 有 700 kDa 的珠子, 进入一个空的, 上限的重力流柱使用1000µL 吸管。将列放置在适当的大小合适的机架上, 安装有一个滴水托盘。</li>
	<li>Uncap 柱底部, 让液体排泄5分钟。一旦排水, 添加10毫升的 PBS, 以洗涤树脂。允许20分钟的洗涤从列排出。</li>
	<li>将浓缩的蛋白酶 K 处理后的样品从步骤3.5 放入15毫升圆锥管中, 并将样品体积增加到5毫升, 用血清吸管添加 PBS。通过摇摆1分钟轻轻地混合管, 然后慢慢地将样品应用到步骤4.2 中用血清学吸管制备的柱上。 注意:一旦去掉柱帽, 样品就会通过该树脂的重力流动;5毫升的样品将完全流经列在10分钟。</li>
	<li>收集流动通过在一个新的15毫升圆锥管。</li>
	<li>使用血清吸管, 再次将收集到的材料应用于树脂, 以除去 700 kDa 以下的剩余材料。</li>
	<li>收集流动通过在一个新的15毫升圆锥管。清洗树脂两次添加1毫升 PBS。从步骤4.5 中收集管道中的流量;最终的总体积将约为7毫升。 注意:在步骤4.6 之后, 样品将被稀释大约35次从原始的样品容量;以下步骤将减少体积, 浓缩纯化的外切样品。</li>
</ol>5. 纯化外来体浓度和总蛋白量化
<ol><li>在使用前, 用 3 kDa 截留分子量过滤器冲洗一个超滤装置, 如步骤3.1 所述。</li>
	<li>从步骤4.6 到超滤装置和离心机在 3700 x g 4 摄氏度的摆动斗转子中添加样品。缓冲区将通过筛选器, 并且可以被丢弃。浓缩外来体将保留在过滤器上方。离心样品, 直到过滤器 (retentate) 上的容积减少到200µL。</li>
	<li>将 retentate 转移到新的离心管。</li>
	<li>用200µL PBS 冲洗滤膜, 吹打膜10次, 从步骤5.3 将洗涤液转移到管子上。这将允许收集任何剩余的外切样本。</li>
	<li>通过添加 PBS, 使样品体积达到500µL。通过缓慢吹打的混合, 上下10次。 注意:协议可以在这里暂停。样品应存放在4°c 隔夜或 20/-80 摄氏度, 长期储存。</li>
	<li>使用制造商的协议, 定量测定样品的蛋白质含量。稀释样品1:10 和1:50 在 PBS。重复运行每个示例。 注意:从血清或血浆总蛋白的10毫克开始, 从步骤5.6 中纯化外来体的总蛋白产量平均为150µg. 如果使用血清或血浆以外的样品, 则可能需要额外的稀释;产量可能因样品类型而异。</li>
</ol>6. 纳米粒子跟踪分析 (NTA) 对外来体的定量和上浆
<ol><li>添加5µg 的纯化外来体 (量化在 5.6) 到1毫升的 PBS 和漩涡十五年代的低中速度。<ol>
<li>将样品放入1毫升一次性注射器中。如果可用, 将注射器设置在自动注射器泵中, 以30µL/分钟的速度注入稀释的外切样品。</li>
		<li>使用视频捕获设置执行 NTA 测量: 屏幕增益为 3-4, 摄像机电平为 12-13。</li>
		<li>定义用于分析的脚本, 以便至少在三十年代使用每个复制的常量流运行三技术复制。对于分析, 请将捕获阈值设置为5。 注意:有关使用自动注射器的详细信息以及视频捕获的设置可在参考13中获得。NTA 的捕获和分析设置必须在不同的示例之间保持一致, 以确保可比性。</li>
	</ol>
</li>
</ol>7. 可溶性蛋白还原的测定
<ol><li>将纯化外来体的 1-10 µg 添加到 SDS 样品缓冲液中, 在 1x MES 中使用 4-12% 双三凝胶进行聚丙烯酰胺凝胶电泳 (页), 在 35 v. 通过应用电压 o 将解析蛋白转移到硝化棉200µm 膜上f 50 V 为 1-1.5 h. 执行西方印迹分析, 评估白蛋白, 载脂蛋白 A 和 B 的存在, 和外切标记 CD 63 遵循标准方法。 注意:7.1 中的方法的完整协议可以在引用14中找到。具体地说, SP007: 聚丙烯酰胺凝胶和 SP011 的运行: 西方的印迹协议。</li>
	<li>用页面和步骤7.1 分开10µg 纯化外来体, 用考马斯染料染色蛋白质带, 遵循标准建议, 以可视化蛋白质变化和样品之间的减少。 注意:对于特定于 CD63 的西方印迹协议, 进一步的细节由迪亚兹et描述。15
</li>
</ol>

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作者没有什么可透露的。

从血液中提高外来体纯度和产量的优化方法, 将提高准确地将细胞外囊泡作为几种疾病的生物标志物来源的能力。目前用于隔离外来体的标准方法, 特别是离心和沉淀方法, 有几个缺点, 包括外切聚合和可溶性蛋白造粒 7, 17, 18。另外, SEC 的使用也显示了外切隔离的显著改善, 保护外来体不被聚合, 并改进了清除常见的污染物非 exosomal 蛋白 9, 11, 18,19. 然而, 如果仅使用 SEC, 载脂蛋白10、更大的囊泡和蛋白质聚合体, 包括白蛋白, 通常与外来体共用。在提出的议定书中, 两个关键步骤有助于克服最后两个提到的限制。首先, 等离子或血清样品在 1.8万 x g 的离心沉淀在样品中的大部分较大的囊泡。其次, 用蛋白酶 K (一种具有广泛特异性的丝氨酸蛋白酶) 预处理血清或血浆样品, 对于降低纯化过程中的白蛋白和载脂蛋白 A-1 和 B 外来体的含量至关重要.我们结合使用蛋白酶 K 与超滤步骤使用的膜与截留分子量 100 kDa 部分洗脱小蛋白质和多肽, 然后我们适应了使用 SEC 树脂与截留分子量 700 kDa 进一步清除小肽/蛋白质从外来体.这个树脂, 最初优选为病毒隔离, 捕获剩余的大蛋白质并且/或者复合物在 700 kDa 之下。隔离的原理, 这 SEC 树脂允许温和恢复的外来体, 因为通过树脂流动的样品是由重力驱动。这样可以减少聚合和外来体的保留完整性, 从而克服基于离心方法的两个主要限制。最后, Baranyai et 。, 在 SEC 处理之前, 等离子体的稀释改进了外切恢复21。在本议定书中, 我们在 SEC 之前加入了稀释步骤, 以增加样品与树脂的接触时间。
该协议的一个重要局限性是蛋白酶 K 处理 exosomal 膜中蛋白质的潜在消化。这可能会损害下游实验, 如果表面暴露的膜蛋白是需要的下游分析, 包括西方印迹, ELISA, 或流式细胞术。为了部分检测蛋白酶 K 对膜相关蛋白的影响, 我们分析了蛋白酶处理样品的存在 CD63, tetraspanin 蛋白, 这是通常存在于外来体。结果表明, 蛋白酶 K 的消化时间和浓度条件的使用并没有导致显著的 CD63 降解。然而, 需要根据特定的研究兴趣进一步评估膜相关蛋白的稳定性。
最后, 所提出的协议比现有的方法具有若干优点。《议定书》中的每一步都表明, 独立研究对外来体的纯度有好处: 更大的囊泡、血清或血浆稀释的造粒, 以及 SEC. 此外, 如上所述, 包括蛋白酶消化步骤, 以消除载脂蛋白和蛋白质聚合, 以及在纯化过程结束时的浓度步骤, 有利于外切的稳定性和产量。最后, 该协议可以很容易地适应, 以隔离外来体从较大的体积样本, 如尿或细胞培养上清液。

外来体是 nanovesicles (大小从30毫微米到150毫微米) 释放的几乎所有的细胞在人体内, 以促进细胞到细胞的通信进程 1, 2.有趣的是, 外来体的组成会根据原始单元格的变化以及单个3、4、5的健康状况而改变.此外, 外来体可以从几种生物液中提取, 如: 唾液、尿液和血液 2.由于这些特点, 外来体被认为是疾病生物标志物的良好来源。不幸的是, 没有标准的方法来隔离外来体。一些实验室考虑多级离心, 以 10万 x g 的最终高速步骤为外切隔离的金标准方法。然而, 最近的研究表明, 离心诱导外来体与可溶性蛋白和其他外来体的聚集, 除了影响外切完整性, 这两个可能会阻碍下游应用程序6, 7.外切隔离的其他常用方法包括但不限于: 以商业聚乙二醇 (PEG) 为基础的试剂、离心超滤和尺寸排除色谱法 (SEC) 沉淀。使用聚乙二醇 (PEG) 聚合物的商业试剂通过使外来体沉淀并形成颗粒而丰富了它们。使用这种聚合物的限制是残留的 PEG 聚合物和大量的可溶性非 exosomal 蛋白在最终产品的污染。超滤利用纤维素膜进行离心纯化浓缩囊泡;外来体保留在过滤器之上, 而较小的杂质和其他蛋白质通过膜 7, 8.就像其他方法一样, 离心超滤由于保留高水平的非 exosomal 蛋白, 包括蛋白质复合物和聚合体, 外来体的净化能力有限。最后, SEC 净化使用多孔树脂分离分子的大小。SEC 已经显示出有希望的结果, 通过捕获大多数的污染物蛋白和保持 exosomal 完整性, 克服了其他方法所遇到的大多数问题, 因为隔离是基于重力或低压系统7, 9。然而, 在 SEC 期间, 大蛋白质集料和脂蛋白的联合分离影响了最终外切制剂的纯度.虽然一些方法已经测试了外切纯化从细胞培养上清液和等离子7, 或仅等离子9, 11, 没有关于直接比较血液的方法的性能信息同一个人的血浆和血清。
在这里, 我们重点研究血液外来体的纯化, 通过比较各种工作流, 以确定是否囊泡浓缩技术是可翻译的血浆和血清。采用纳米微粒跟踪分析和印迹方法, 对最终产物中外切浓度、纯度和蛋白质组成的差异进行量化。最后的方法, 详细的在本协议中, 增加囊泡数和减少非 exosomal 蛋白水平。重要的是, 大量减少常见的共沉淀蛋白, 包括白蛋白和载脂蛋白。这两种蛋白质在血液中的高丰度以及它们与外来体共同纯化的频率导致了 "纯化" 样品之间的不一致, 从而扭曲了下游的分析结果。该协议包括使用商业上可用的 SEC 树脂;该树脂是由多孔珠组成, 其中蛋白质小于 700 kDa 可以进入珠。一旦内, 蛋白质由辛胺配体保留。洗脱液由外来体和大于700kDa 12 的分子组成。此外, 为了减少载脂蛋白的蛋白质聚集和抑制, 我们在工作流中加入了蛋白酶消化步骤。目前, 还没有单一的技术能够最大限度地提高外切产量, 同时减少共纯化非 exosomal 蛋白。本研究表明, 采用蛋白酶消化预处理与多种回收纯化相结合的纯化方法, 可提高血清和血浆外切的产量和纯度。

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protein digestion, ultrafiltration, and size exclusion chromatography to optimize the isolation of exosomes from human blood plasma and serum

外来体是一种从所有细胞类型中释放出来的 nanovesicle, 可以从任何体液中分离出来。外来体的内容, 包括蛋白质和 rna, 对它们所派生的细胞是独一无二的, 可以作为疾病的指标。一些常见的浓缩协议, 包括离心, 产外来体可溶性蛋白污染物。具体地说, 我们发现血液中最丰富的蛋白质经常与外来体共同纯化, 并可能混淆下游蛋白质组研究, 挫败低丰度生物标志物候选者的识别。另外的关注是由于非 exosomal 蛋白水平的不一致表示, 外切蛋白量化 irreproducibility。这里详述的协议是为了去除与外来体共纯化的非 exosomal 蛋白, 增加了外切纯化过程的严格。采用五例献血者配对血浆和血清对五种方法进行比较。用纳米微粒跟踪分析和微二辛可宁酸蛋白检测结果表明, 采用超滤和粒径排除色谱相结合的复合协议, 可获得最佳的囊泡浓缩和可溶性蛋白去除。西方印迹被用来验证预期丰富的血液蛋白, 包括白蛋白和载脂蛋白, 已耗尽。

以下数据是使用五健康献血者配对血浆和血清样本获得的。为了确定各处理步骤的效果, 对所提出的协议进行了五种变异, 并对外切恢复和非外切蛋白去除进行了比较。方法全身扫描仪最包括: 1) 秒 700 kDa + 超滤 3 kDa;2) 超滤 100 kDa;3) 蛋白酶 K + 超滤 100 kDa;4) SEC 700 kDa + 超滤 100 kDa, 5) 蛋白酶 K + 超滤 100 kDa + 秒 700 kDa + 超滤 3 kDa。
总的来说, 涉及 SEC 700 kDa (1、4和 5) 的所有方法在样本、血清和血浆 (图 1A-B) 中, 每微克蛋白质的囊泡数量显著增加。但是, 方法5比血清 (图 1C) 产生的血浆每微克的囊泡数量明显增加。反之, 在使用 SEC 700 kDa 方法 (1、4和 5) 时, 纯化外切样品中的蛋白质浓度明显降低。单向方差分析和 Tukey 的多项比较试验表明, 方法2和3后的最终蛋白质浓度显著高于方法1、4和 5 (p < 0.001) 后的最终蛋白质浓度。差异是一致的使用血浆或血清, 然而, 与方法1纯化外来体的最终蛋白浓度显著高于 (t 检验, p < 0.05) 时使用血清 ( 图2A)。用不同的五种方法对纯化外来体的蛋白质分布进行评价, 在一页中解决了10µg 样品, 并与考马斯染料染色。结果表明, 从方法2和3中的 "纯化外来体" 中的大多数蛋白质是由强频带 ~ 65 kDa (图2B) 表示的白蛋白。
下一步是根据白蛋白的存在, 血液中最丰富的蛋白质来确认孤立外来体的纯度增加。超滤过程显示, exosomal 分数的白蛋白显著浓度, 部分减少了前处理样品与蛋白酶 K (图 3A, 车道 S 和 P)。在该过程中加入 sec 700 kDa 减少了白蛋白的数量 (图 3A、车道 P1/S1 和 P4/S4), 但 sec 700 kDa、蛋白酶 K 和超滤的组合表明, 从纯化的外来体中清除白蛋白的效率最高 (图 3A, 车道 P5/S5)。最近的研究表明, 在外切纯化过程中, 载脂蛋白也通常是相互隔离的10, 16.具体地说, ApoB, 这是存在于低密度脂蛋白, 被发现高度集中在纯化外来体从人类血液 10.因此, 我们评价了脂蛋白 ApoB 和 ApoA-1 的共同分离。离心方法和离心和 SEC 700 kDa 的结合表明, 与血清和血浆中检测到的血量 (图 3B、车道 P1/S1、P2/S2 和 P4/S4) 相比, ApoB 的数量也相似;补充图 1/2)。有趣的是, 蛋白酶 K 的加入导致了 ApoB 从纯化样品中完全降解 (图 3B、车道 P3/S3 和 P5/S5)。同样, ApoA-1, 主要蛋白质成分的高密度脂蛋白在人血浆, 是减少了所有方法, 包括 SEC 700 kDa 或蛋白酶 K (图 3C)。虽然像白蛋白一样, 100 截留分子量超滤本身无法显著减少共纯化 ApoA-1 的数量。
最后, 为了确定蛋白酶 K 对外来体外膜蛋白的影响, 我们评价了外来体 CD63 tetraspanin 的存在。对西方印迹的分析表明, 该蛋白不仅可在使用蛋白酶 K (图 3D、车道 P3/S3 和 P5/S5) 的方法中检测到, 而且使用方法5的 CD63 带具有更强烈的信号 (图 3D、车道 P5/S5)。这一发现表明, 方法5的结果要么是较高浓度的外来体或增加 CD63 抗体结合, 由于蛋白质聚集减少与外切表面。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57467/57467fig1.jpg"> 图 1: 外切五种不同纯化方法后从血浆和血清中产生.(A) 从血浆中获得的外来体浓度 (n = 5)。(B) 从血清中获得的外来体浓度 (n = 5)。(C) 从5配对血浆和血清中外来体每微克蛋白质的数量之间的比较。x 轴上的数字表示所使用的每个方法, 1 = 秒 700 kDa + 超滤 3 kDa;2 = 超滤 100 kDa;3 = 蛋白酶 K + 超滤 100 kDa;4 = 秒 700 kDa + 超滤 100 kDa, 5 = 蛋白酶 K + 超滤 100 kDa + 秒 700 kDa + 超滤 3 kDa。在 A 和 B 中, 通过单向方差分析和 Tukey 的多比较测试来计算差异。在 C 中, 用 t 检验法计算血清和血浆的差异。*p < 0.05;误差线: 95% 的置信区间。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57467/57467fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57467/57467fig2.jpg"> 图 2: 用五种不同的方法从血浆和血清中提取纯化外来体的蛋白质。(A) 从血浆和血清中获得的纯化外来体中总蛋白产量的比较 (n = 5)。(B) 考马斯染色的10µg 纯化外来体的代表性图片, 由 SDS 页解决;n = 1, 配对血浆和血清。数字在图代表每个方法, 1 = 秒 700 kDa + 超滤 3 kDa;2 = 超滤 100 kDa;3 = 蛋白酶 K + 超滤 100 kDa;4 = 秒 700 kDa + 超滤 100 kDa, 5 = 蛋白酶 K + 超滤 100 kDa + 秒 700 kDa + 超滤 3 kDa。在A中, 通过 t 检验计算血浆和血清衍生样品之间的差异。*p < 0.05。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57467/57467fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57467/57467fig3.jpg"> 图 3: 减少从人血液中分离出来的外来体的正常污染物和标志性蛋白 CD63 的稳定性。西部污点评估: (A) 白蛋白, 1 µg 每车道;(B) 载脂蛋白 B, 每车道1µg;和 (C) 载脂蛋白 A1, 每车道10µg;(D) CD63, 每车道1µg。L = 梯子;S = 粗血清;P = 粗等离子体。纯化外切: 采用5种不同的方法进行纯化。数字在图代表每个方法, 1 = 秒 700 kDa + 超滤 3 kDa;2 = 超滤 100 kDa;3 = 蛋白酶 K + 超滤 100 kDa;4 = 秒 700 kDa + 超滤 100 kDa, 5 = 蛋白酶 K + 超滤 100 kDa + 秒 700 kDa + 超滤 3 kDa。有代表性的结果从配对血清和血浆的一个病人;捐助者样本之间的趋势是一致的。蛋白质负载随印迹变化;规范化是基于对总样本浓度的测定。每条车道的总蛋白质负荷为每个污点指定。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57467/57467fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充图 1: 原始 uncropped 西部污点从图 3.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57467/Supplement1.tif" target="_blank">请单击此处查看此图的较大版本.</a>
补充图 2: 白蛋白、ApoB、ApoA-1 和 CD63 带的密度量化图 3;使用了 ImageJ 软件。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57467/Supplement2.tif" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum at JoVE.com

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

在这里, 我们提出了一个协议, 以净化外来体从血浆和血清与减少共同纯化的非 exosomal 血液蛋白。优化的协议包括超滤、蛋白酶处理和尺寸排除色谱。增强纯化外来体的好处下游分析, 包括更准确的定量的囊泡和蛋白质组特征。

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 ...

protein-digestion-ultrafiltration-size-exclusion-chromatography-to

Nanyang Technological University

Research

JoVE Journal

Biochemistry

16.4K 次观看.  Colorado State University. 在这里, 我们提出了一个协议, 以净化外来体从血浆和血清与减少共同纯化的非 exosomal 血液蛋白。优化的协议包括超滤、蛋白酶处理和尺寸排除色谱。增强纯化外来体的好处下游分析, 包括更准确的定量的囊泡和蛋白质组特征。

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

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

在线体积排阻和离子交换色谱法SAXS光束线

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

HEK 细胞 Mitoribosome 的快速分离

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

人类外周血神经细胞分离通过评估Rab10磷酸化来询问帕金森氏症相关的LRRK2激酶途径

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

离子交换色谱 (IEX) 与多角度光散射 (ALS) 相结合, 用于蛋白质分离和表征

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

蛋白质的表征(按尺寸-排除色谱与多角度光散射相结合(SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps &#160;of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

Presented is a protocol to fabricate a paper-based device for the effective enrichment and isolation of microvesicles and exosomes.

微量和外显子的纸基预集中和分离

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they ...

Analysis and Specification of Starch Granule Size Distributions

Starch from all plant sources are made up of granules in a range of sizes and shapes having different occurrence frequencies, i.e., exhibiting a size and a shape distribution. Starch granule size data determined using several types of particle sizing techniques are often problematic due to poor reproducibility or lack of statistical significance resulting from some insurmountable systematic errors, including sensitivity to granule shapes and limits of granule-sample sizes. We outlined a procedure for reproducible and statistically valid determinations of starch granule size distributions using the electrical sensing zone technique, and for specifying the determined granule lognormal size distributions using an adopted two-parameter multiplicative form with improved accuracy and comparability. It is applicable to all granule sizing analyses of gram-scale starch samples, and, therefore, could facilitate studies on how starch granule sizes are molded by the starch biosynthesis apparatus and mechanisms; and how they impact properties and functionality of starches for food and industrial uses. Representative results are presented from replicate analyses of granule size distributions of sweetpotato starch samples using the outlined procedure. We further discussed several key technical aspects of the procedure, especially, the multiplicative specification of granule lognormal size distributions and some technical means for overcoming frequent aperture blockage by granule aggregates.

Presented here is a procedure for reproducible and statistically valid determinations of starch granule size distributions, and for specifying the determined granule lognormal size distributions using a two-parameter multiplicative form. It is applicable to all granule sizing analyses of gram-scale starch samples for plant and food science research. &#160;

淀粉颗粒大小分布的分析和规格

Starch from all plant sources are made up of granules in a range of sizes and shapes having different occurrence frequencies, i.e., exhibiting a size and ...