A.J.C、J.A.T 和 I.L 通过 Horizon 2020 项目 ACEnano（授予 No.H2020-NMBP-2016-720952）。W.Z承认中国奖学金理事会（CSC，第201507060011号）的博士学位资助。R.R承认荷兰科学研究组织维迪赠款计划的财政支持（NWO Vidi 723.0160330）。

根据莱顿大学和因斯布鲁克医科大学的IRB协议指南，人类生物流的使用得到批准。在调查人类或动物生物流体时，需要获得研究机构的伦理批准，并且已获得本协议中所示的结果。此外，亦应进行适当的报告，以确保数据在未来的工作9、29、30的透明度和可重复性。
1. 背景电解质的制备
注:所有溶剂应在合适的烟气罩中准备，所有步骤（实验室涂料、手套和护目镜）都必须使用足够的个人防护设备。在每一步中，都需要低结合塑料瓶，以尽量减少从玻璃器皿中浸出的盐对样品的分析损失和污染。
<ol><li>每天准备 10% 醋酸 BGE （v/v），pH 2.2 用于代谢学。<ol>
<li>进入10mL体积的烧瓶中加入1mL的醋酸，然后在标记的生产线上加入去离子（DI）水并彻底混合。</li>
	</ol>
</li>
	<li>准备100mM醋酸，pH 2.9每天的基础上，为蛋白真学。<ol>
<li>进入10mL体积的烧瓶中加入57微升醋酸，然后加入DI水到标记线和彻底混合。</li>
	</ol>
</li>
</ol>2. NM 准备
<ol><li>使用已发布的指南31、32、33大力分散水中的NM。 注:在制定本协议时，使用了7个NM，分别是100和1000纳米碳基聚苯乙烯用于蛋白质和代谢物日冕。100 nm 未修改的聚苯乙烯 NMs，13 nm anatase TiO2 NM 未修改或涂有聚丙烯酸酯或 PVP 聚合物，22 nm 未修改 SiO2 Nms 用于蛋白质和代谢物日冕。虽然该方法是使用这些 NM 开发和演示的，但应该指出，这种方法将适用于各种 NM 成分、大小、形状和形态。</li>
	<li>使用动态光散射34、35、单粒子电感耦合等离子质谱36、纳米粒子跟踪分析37，或利用zeta sizer34传输电子显微镜和表面电荷作为zeta电位，描述粒子大小。</li>
</ol>3. 生物分子日冕形成
<ol><li>将2mL的人类血浆（或选择的生物流体）分成1兆瓦，一个用于代谢物和蛋白质日冕，第二个用于等离子体代谢物的表征。</li>
	<li>在 37 °C 下在人体等离子体中孵育 1 毫克/毫升的 NM，同时在热混合器中以 500 rpm 轻轻混合。孵化后离心机样品在 4，000 x g 15 分钟在 4 °C 颗粒 Nms.</li>
	<li>收集等离子体超高纳剂进行代谢物日冕分析。保留 NM 蛋白日冕复合物，用于蛋白质日冕表征。</li>
</ol>4. 蛋白质日冕分离
<ol><li>将 NM 蛋白复合物（颗粒）在 1 mL 的 10 倍 PBS 缓冲器和漩涡中大力重复 2 分钟，以去除未绑定的蛋白质。将 PBS 溶液在 4，000 x g 下离心，在 4 °C 下 15 分钟将 NM 颗粒化，并小心地取出超高颗粒，而不会干扰颗粒。</li>
	<li>将颗粒以 1 mL 的碳酸氢铵缓冲器 （ABC） （100 mM， pH 8.0） 和漩涡强烈地重复 2 分钟，以去除未受约束的蛋白质和不受欢迎的盐。离心机在 4，000 x g 15 分钟在 4 °C 颗粒 Nms;取出并丢弃超高等。重复此步骤。</li>
	<li>通过溶解含有 10 mM 二乙醚的 ABC 缓冲器 （100 mM pH 8） 中的 NM 蛋白颗粒，减少蛋白质脱硫键。在 56 °C 下将减少解决方案孵化 30 分钟。</li>
	<li>要消化蛋白质，请添加 2 μg 的测序级 Trypsin 到 20 μL 的 ABC 含有 0.1% 表面活性剂。在 37 °C 下将消化液孵化 16 小时。</li>
	<li>在 100 mM ABC 中加入 55 mM 的 20 微升碘酰胺，并在室温下孵育 20 分钟，从而对样品进行碱化。</li>
	<li>加入 20 μL 的 0.1 M HCl 以切割表面活性剂，并在室温下离开 10 分钟。</li>
	<li>使用 C18 包装脱盐移液器尖端丰富和脱盐肽。<ol>
<li>用 80 μL 0.1% 的甲酸 50% 丙酮三聚醇润滑尖端 5 次， 用 80 μL 0.1% 的甲酸清洗 5 次，绘制和提取样品 10 次，通过冲洗 80 μL 0.1% 的甲酸 5 次，用 80 μL 0.1% 的甲基苯丙酸将样品提取到干净的低结合 PCR 管中。</li>
	</ol>
</li>
	<li>将样品放样并储存在 -20 °C，直到分析。</li>
</ol>5. 代谢物日冕分离
<ol><li>从第 3.3 步从 NM 等离子体孵化中取出控制等离子体和超高纳特，并在样品准备过程中保持冰上。</li>
	<li>将每个瓶中的 50 μL 放入单独的瓶中，用 DI 水稀释十中 1。取每个稀释样品的 50 μL，然后移动到新的小瓶进行第 5.3 步。</li>
	<li>加入 200 μL 氯仿、250 μL 甲醇和 350 μL DI 水，大力涡流混合物 2 分钟。离心机 10 分钟，在 4 °C 下 20，800 x g。</li>
	<li>服用 500 μL 超高超纳坦，在 4 °C 下通过 3 kDa 离心过滤器过滤 2 小时，10，000 x g。 服用 430 μL 的超滤和干燥的速度真空。样品现在可以在分析前冻结。</li>
</ol>6. 编制CESI-MS系统38
注:在安装毛细血管之前，请检查毛细血管的入口和出口端是否完好无损，毛细血管中是否存在可见的破损。
<ol><li>安装中性涂层毛细细丝蛋白质日冕分析39。<ol>
<li>按照制造商指南，将新的中性涂层毛细细圈（90 厘米长，内部直径为 30 μm）放入 CESI 仪器中。</li>
	</ol>
</li>
	<li>准备 CESI 毛细血管进行蛋白化分析。<ol>
<li>在 100 psi 下使用 100 mM 的 HCl 朝前方方向冲洗分离毛细管 5 分钟，并在毛细管出口处检查水滴形成。冲洗分离毛细管与 BGE 在 100 psi 10 分钟，然后 DI 水在 100 psi 为 30 分钟（这只需要新的毛细血管）。</li>
		<li>将分离毛细管和导电毛细管与 BGE 在 90 psi 下冲洗 5 分钟，并确保两个毛细血管出口端形成液滴。</li>
		<li>用 DI 水在 90 psi 下冲洗分离毛细管 10 分钟，然后用 0.1 M HCl 冲洗 50 psi 10 分钟，然后用 DI 水冲洗 90 psi 10 分钟，最后用 90 psi 冲洗 10 分钟。将CESI与MS结合使用纳米喷雾源和适配器，如前所述38。</li>
	</ol>
</li>
	<li>安装裸熔二氧化硅毛细细用于代谢物日冕特征38。<ol>
<li>按照制造商的指导原则，将新的裸熔硅毛细细（90 厘米长，内部直径为 30μm）放入 CESI 仪器中。</li>
	</ol>
</li>
	<li>为代谢分析准备 CESI 毛细血管。<ol>
<li>在 50 psi 下使用 100% MeOH 朝前方方向冲洗分离毛细管 15 分钟，并在毛细管出口处检查水滴形成。将分离毛细管和导电毛细管与 BGE 在 90 psi 下冲洗 5 分钟，并确保两个毛细血管出口处形成液滴。</li>
		<li>用 DI 水在 90 psi 下冲洗分离毛细管 10 分钟，然后用 0.1 M NaOH 冲洗 50 psi 10 分钟，然后用 DI 水冲洗 90 psi 10 分钟，最后用 90 psi 冲洗 10 分钟 BGE。使用之前描述的纳米喷雾源和适配器将 CESI 与 ESI-MS 耦合。38
</li>
	</ol>
</li>
</ol>7. 用于蛋白质日冕分析的 CE-MS
<ol><li>从步骤 4.8 中重新恢复样品，在 20 μL 的 50 mM 醋酸铵 pH 4.0 中。</li>
	<li>执行 5 psi 10s 流体动力学样品注射。使用 30 kV 分离电压与以下压力梯度分开:1 psi 0-10 分钟，1.5 psi 10-35 分钟，5 psi 使用 100 mM、pH 2.9 醋酸作为 BGE 35-45 分钟。</li>
	<li>收集 250-2000 m/z 之间的质谱，并获取前 10 名数据依赖性碎片采集。</li>
	<li>在样品之间，用0.1 M HCl冲洗毛细管，3分钟100 psi，10分钟100 psi BGE分离毛细管，3分钟100 psi用于导电液毛细管。</li>
</ol>8. 用于代谢物日冕分析的 CESI-MS
注:所有样品必须分析两次，一次为正模式检测阴离子，一次为阴性模式检测阴离子。控制等离子体样品至少需要分析5次。
<ol><li>在 430 μL 的 DI 水中从第 5.4 步中重新使用 NM 暴露和未暴露的等离子体样本，并大力涡流 2 分钟。通过 0.1 μm 膜过滤器过滤。</li>
	<li>服用 95 μL 的过滤液，在 200 μM 的阴离子（L-甲氨基硫酮）和 400 μM 的离子（2，2，4，4，4-D4-柠檬酸）和涡流中加入 5 μL 的内部标准。在 CESI-MS 分析之前，在 4°C 下 4，000 x g 的离心机 10 分钟。</li>
	<li>在 2 psi 下以水动力学注射样品，用于 30s（cations）和 40s（离子）。</li>
	<li>在10%醋酸中分离代谢物，在前进（离子）和反向（离子）模式下分离电压为30千伏。反向分离模式辅助分离毛细管的 0.5 psi 向前压力。</li>
	<li>设置质谱仪，以正（离子）或负（离子）模式收集质量范围为 65-1000 m/z 的数据。 在样品之间，用 0.1 M HCl、0.1 M NaOH、DI 水和 BGE 在 50 psi 冲洗毛细管 2 分钟。</li>
</ol>

J.A.T 是 AB Sciex 英国有限公司的一名员工，作为行业合作伙伴参与 ACEnano 项目，J.A.T 和所有其他作者在这项工作中没有利益冲突。

这是第一个使用CESI-MS来描述完整的生物分子日冕，结合蛋白质和代谢物的方法。能够从同一样本中描述蛋白质和代谢物，将显著增加从单个实验暴露中收集的数据量，从而对日冕形成过程及其对纳米药物的NMs毒性和功效的影响有新的见解。此外，CESI-MS 是一个理想的平台，只需更换毛细头，就可描述这两类生物分子的特点。展望未来，为非无性CE开发的新方法将使脂质学能够使用CE-MS40 进行，因此现在可以使用单一平台分析蛋白质、代谢物和脂质。目前的传统方法需要两个专门的LC-MS平台，一个用于蛋白质日冕，一个用于代谢物日冕。因此，这种方法可以使用单个分析平台收集两倍的数据。
对于这种方法，特别是代谢物日冕有一些注意事项，因为本协议建议间接测量日冕。因此，当代谢物水平相对于 NM 存在高浓度且随后基质中代谢物浓度下降较小时，可能会出现问题。然而，与蛋白质日冕不同，由于每个NM都有独特的表面化学成分，因此不太可能开发出"一刀切"的方法来分离代谢物日冕。今后，将更有可能开发和验证NM分离代谢物日冕的具体方法:这将仅适用于特定的 NM。尽管如此，CESI-MS仍将是极地带电代谢物的定性非常合适的平台，无论它们是如何被分离的。同样值得注意的是，如果在设计用于给 NM 弹丸的离心步骤中不形成颗粒，则可能需要更高的离心力:这最有可能发生在密度较低的 Nms 。同样重要的是，由于毛细血管孔窄，所有过滤步骤都遵守协议，如果样品中未充分消除任何颗粒物，这些毛细血管很容易被阻塞。
虽然蛋白质日冕多年来一直是一个紧张的研究领域，但将这种能力与代谢物日冕相结合的能力是科学家研究生物纳米界面6、14的新领域。迄今为止，这些研究大多集中在代谢物日冕9，28的非极性，脂质部分。此当前方法可对日冕中高极性和带电代谢物进行表征，其中包括参与能量代谢、糖解和 DNA 合成的重要代谢物。因此，当与蛋白术工作流程相结合时，生物分子日冕的更整体特征是可能的。这有助于更深入地分析生物纳米相互作用的发展。该方法通过蛋白质和代谢物与膜受体的相互作用，增强了对受体介质介质内分泌的机械洞察。此外，可以探讨蛋白质和小分子之间的日冕内部相互作用:例如，最近已经表明，日冕中的蛋白质在代谢物15的招募中起着关键作用。值得注意的是，图3和图4中的代表性结果是代谢物的绝对定量，然而，使用多变量数据分析来比较对照组中与暴露等离子体相比的所有CE-MS特征的非目标方法也可以阐明代谢物的结合。因此，有一个重要的范围来研究代谢物和蛋白质如何以及哪些影响它们各自的招募到NM日冕。这些额外的研究可能有助于设计日冕，以优化纳米药物的输送，或发现进一步的障碍，如抑制药物行动，因为生物分子日冕阻塞或掩盖靶向功能。
CESI-MS 的纳米规模流量约为 20 nL/min，有机溶剂的使用最少，在溶剂使用方面，绿色和经济凭据分别比标准 LC-MS 和纳米 LC-MS 有所提高，这是代谢学和蛋白造影学研究面临的主要挑战。CESI-MS为迁移时间和高峰区域提供了高度可重复的结果，与基于LC-MS的方法11，15相比，效果优于。此外，与纳米LC-MS相比，结搬在CESI-MS中不是问题。因此，样品分析之间不需要进行广泛的系统清理，这大大提高了样品吞吐量11。
总之，拟议的工作流程是首次详细说明对 NMs 完整生物分子日冕的蛋白质和代谢物成分进行特征化的方法。这开启了生物纳米相互作用的新方面，即代谢物日冕，从而能够从同一样本中收集两倍的数据，从而更全面地了解生物纳米界面的相互作用。

生物纳米相互作用位于纳米材料表面与生物分子吸附到NM的生物母体之间的界面上，是支撑纳米安全性和纳米医学1的一个密集研究领域。这一层吸附的生物分子为NMs提供生物识别，因此细胞将其识别为"自我"，从而影响细胞吸收、分布和生物终点2、3、4。绝大多数的生物纳米研究都集中在日冕内的蛋白质上，并证明不同成分的蛋白质在数量和质量上各不相同。这种变化取决于NM的特性，如大小，功能化和材料，以及生物基质的特性，包括成分和盐含量5。生物纳米界面的一个日益浓厚的兴趣领域是吸附到NMs6的代谢物。一些研究表明，与蛋白质一样，NM特性是决定日冕中代谢物成分的关键因素，它们可以影响NM暴露6、7、8的生物结果。
在生物分子日冕的研究中，其特征、日冕形成、日冕内生物分子的分离、通过定性或定量质谱和识别检测有四个不同的阶段。迄今为止，已经采用了一系列技术来分离蛋白质日冕，包括在SDS-PAGE运行缓冲器中沸腾、溶剂和盐提取或直接消化在NM表面原位的蛋白质。就日冕中的代谢物而言，使用溶剂的各种方法，甚至作为解决方案8、10、11提出的NMs的溶解，隔离更为复杂。然而，与蛋白质日冕不同，"一刀切"的方法不太可能适用于代谢物与NM的分离，因为代谢层占据了广阔的化学空间和可能的NM的多样性。另一种方法是将NM暴露前后的生物基质与代谢物浓度的差异描述为NS12。
这种替代方法将适用于所有生物流体和 NM，虽然它确实需要两倍的分析，因此它提供了代谢物日冕的更精确的测量，并且没有从 NM 表面回收低代谢物被误认为是特定代谢物的低结合的风险。
生物分子日冕分析的第二步是生物分子的检测。传统上，对于日冕中的蛋白质来说，这是纳米LC-MS的职权范围，纳米LC-MS是蛋白质组学的主力:其他方法，如NMR13，1D和2D SDS-PAGE也已应用。在代谢物日冕LC-MS7方面，利用GC-MS8和直接输液质谱法14。然而，一种新的方法最近开始获得牵引力，即毛细管电泳-质谱学（CE-MS）11，15，并已在NM实验室作为一种独立的技术，以描述NMs16的各种物理和化学性质。CE-MS是纳米LC-MS和GC-MS的正交分离技术，能够检测高极和带电代谢物17，18。此外，CE-MS非常适合分析蛋白质及其后翻译修改，如磷酸化和糖化19，20，21，22。最后一步，身份识别和数据分析可以通过多种方式进行，具体取决于是否使用了定量/定性或目标/非目标方法，但是，这不属于本协议的职权范围。
CESI-MS 是 CE 和纳米电子接口的结合体，是 CE 技术利用无护套接口的最新进步。这使得超低流量CE（<20 nL/min）与高分辨率质谱仪直接连接，无需稀释，从而显著提高检测灵敏度23，24，25。CESI-MS 使体积有限的样品（< 10 μL） 进行分析，同时保持高度敏感的分析26。CESI-MS还优于传统的低流速纳米LC-MS方法，用于蛋白造学和代谢学的可重复性27，28，吞吐量，并延续，使其成为一个令人兴奋的前景，为完整的生物分子日冕表征。
为了突出CESI-MS在生物纳米相互作用分析方面的潜力，本工作描述了从单个人类等离子体样本中分离NM生物分子日冕所需的样品制备，以便最大限度地利用来自完整生物分子NM日冕蛋白质和代谢物方面的数据。当我们报告人类血浆的使用时，这个协议同样适合其他生物流体，如血清、完整的细胞培养介质、细胞解冻液、脑脊液或尿液。然后，将使用中性涂层的CESI毛细血管对蛋白质日冕和光秃秃的二氧化硅毛细血管进行分析，用于青贮和电离代谢物。

<table><tbody><tr><td>1,4-Dithiothreitol</td><td>Sigma</td><td>10197777001</td><td></td></tr><tr><td>2,2,4,4-D4-citric</td><td>Simga</td><td>485438-1G</td><td>Internal standard anion</td></tr><tr><td>Ammonium Acetate &gt;98%</td><td>Sigma</td><td>A1542</td><td></td></tr><tr><td>Ammonium Bicarbonate &gt;99.5%</td><td>Sigma</td><td>09830</td><td></td></tr><tr><td>Capillary cartridge coolant</td><td>Sciex</td><td>359976</td><td></td></tr><tr><td>CESI 8000 instrument</td><td>Sciex</td><td>A98089</td><td></td></tr><tr><td>CESI Cap</td><td>Sciex</td><td>B24699</td><td></td></tr><tr><td>CESI Vials</td><td>Sciex</td><td>B11648</td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>650498</td><td>Toxic, use in a fume hood</td></tr><tr><td>Glacial Acetic acid</td><td>Sigma</td><td>A6283</td><td></td></tr><tr><td>Hydrochloric acid 1mol/L</td><td>Sigma</td><td>43617</td><td></td></tr><tr><td>Iosoacetamide</td><td>Sigma</td><td>I1149</td><td></td></tr><tr><td>L-methionine sulfone</td><td>Sigma</td><td>M0876-1G</td><td>Internal standard cation</td></tr><tr><td>Methonal (LC-MS Ultra Chromasolve)</td><td>Sigma</td><td>14262</td><td>Use in a fume hood</td></tr><tr><td>Microvials</td><td>Sciex</td><td>144709</td><td></td></tr><tr><td>nanoVials</td><td>Sciex</td><td>5043467</td><td></td></tr><tr><td>Neutral Surface Cartridge 30 &amp;micro;M ID x 90 cm total length</td><td>Sciex</td><td>B07368</td><td></td></tr><tr><td>OptiMS Adapter for Sciex Nanospray III source</td><td>Sciex</td><td>B07363</td><td>B07366 and B83386 for Thermo mass spectrometers, B85397 for Waters mass spectrometers, B86099 for Bruker mass spectrometers</td></tr><tr><td>OptiMS Fused-Silica Cartridge 30 &amp;micro;m ID x 90 cm total length</td><td>Sciex</td><td>B07367</td><td></td></tr><tr><td>Phospahte buffered saline 10x</td><td>Sigma</td><td>P7059</td><td></td></tr><tr><td>Pierce C18 Tips, 100 &amp;micro;L bed</td><td>Thermo Fisher Scientific</td><td>87784</td><td></td></tr><tr><td>Polystyrene 100 nm</td><td>Polysciences Inc</td><td>876</td><td></td></tr><tr><td>Polystyrene carboxylated 100 nm</td><td>Polysciences Inc</td><td>16688</td><td></td></tr><tr><td>Polystyrene carboxylated 1000 nm</td><td>Polysciences Inc</td><td>08226</td><td></td></tr><tr><td>Rapigest SF (surfactant)</td><td>Waters</td><td>186001860</td><td></td></tr><tr><td>Sequencing Grade modified Trypsin</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>SiO2 Ludox TM-40</td><td>Sigma</td><td>420786</td><td></td></tr><tr><td>Sodium Hydroxide solution</td><td>Simga</td><td>72079</td><td></td></tr><tr><td>TiO2 Dispex coated</td><td>Promethion particles</td><td>Bespoke particles</td><td></td></tr><tr><td>TiO2 PVP coated</td><td>Promethion particles</td><td>Bespoke particles</td><td></td></tr><tr><td>TiO2 uncoated</td><td>Promethion particles</td><td>Bespoke particles</td><td></td></tr></tbody></table>

capillary electrophoresis mass spectrometry approaches for characterization of the protein and metabolite corona acquired by nanomaterials

过去十年来，生物分子从周围的生物母体吸附到纳米材料（NM）表面形成日冕一直引起人们的兴趣。对生物纳米界面的兴趣源于生物分子日冕赋予NM一个生物特性，从而导致身体识别它们为"自我"。例如，先前的研究表明，日冕中的蛋白质能够与膜受体相互作用，从而影响细胞吸收，并确定日冕负责细胞贩运的NMs及其最终的毒性。迄今为止，大多数研究都集中在蛋白质日冕上，忽略了日冕中所含代谢物的可能影响，或整个生物分子日冕中各成分之间的协同效应。因此，本研究演示了利用自下而上的蛋白质组学和代谢方法同时描述生物分子日冕的蛋白质和代谢物成分的方法。这包括使用用于增加蛋白质恢复的表面活性剂的蛋白质日冕的粒子上摘要，以及通过分析 NM 暴露前后的代谢物矩阵对代谢物日冕的被动特征。这项工作引入毛细电电泳 - 质谱学 （CESI-MS） 作为 NM 日冕特征的新技术。这里概述的协议演示了如何使用CESI-MS对NMs获得的蛋白质和代谢物日冕进行可靠的定性。转到 CESI-MS 可大大减少所需的样品体积（与传统的液相色谱 - 质谱仪 （LC-MS） 方法相比），可从 5 μL 的样品中多次注射，使其成为体积有限的样品的理想选择。此外，由于 CESI-MS 的低流量（< 20 nL/min）以及水解质的使用，消除了对有机溶剂的需求，因此分析对 LC-MS 的环境影响有所降低。

所述方法是首次使用相同的人类血浆样本来描述NM生物分子日冕中的蛋白质和代谢物。该方法能够检测>200种蛋白质和>150代谢物，从而能够确定完整的生物分子日冕的最全面概述，从而能够更好地了解NMs细胞附着、吸收和影响。
CESI-MS用于蛋白体学（图1）和代谢（图2）分离和检测，表明每种方法的两个毛细血管都有良好的分离窗口。
这种方法能够区分日冕中蛋白质在广泛的NM成分，从而证明该方法在广泛的物理和化学特性（表1）中对NM的适用性。
代谢物日冕的独特指纹也可以使用这种方法的代谢分支（图3）来区分。虽然这种方法利用一种被动的方法来描述NM代谢物日冕，但它仍然能够揭示对代谢物在生物分子日冕中的作用的有趣见解，如异位体的微分吸附（图4）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61760/61760fig01.jpg"> 图1:在粒子消化后，使用CESI-MS使用SI-MS进行SI2蛋白日冕分离的SiO2蛋白日冕分离的表例电球图。<a href="https://www.jove.com/files/ftp_upload/61760/61760fig01large.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/61760/61760fig02.jpg"> 图2:CE-MS从等离子体内源化合物中提取的离子电极图示例。 编号化合物如下: 1. 兽人:2. 莱辛;3. 精氨酸;4. 赫斯蒂丁:5. 肌酸:6. 甘氨酸;7. 阿兰宁;8. 虚线:9. 伊索卢辛:10. 莱辛;11. 塞林;12. 三甲宁;13. 芦笋;14. 甲氨酸;15. 谷氨酰胺;16. 谷氨酸;17. 苯-D5-阿兰宁;18. 苯丙氨酸;19. 泰罗辛;20. 临线;21. 硫化甲酸二甲酸（内部标准）。在开放访问下发布创意共享 CC BY 许可证，并转载自张等人17。 <a href="https://www.jove.com/files/ftp_upload/61760/61760fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
表1:对6个NMs阵列的蛋白质日冕分析。在二氧化硅（SiO2）、钛（TiO2）、PVP封顶钛（TiO 2-PVP）、迪斯封顶钛（TiO2-Dispex）、聚苯乙烯和碳化聚苯乙烯NMs上识别的蛋白质类的热图。 百分比是指蛋白质相对于每种蛋白质前3个肽丰度的总蛋白质含量的丰度。鉴于在不同NM日冕中观察到的蛋白质相对丰度存在差异，很明显，这个提议的协议对于区分不同NM的蛋白质日冕非常敏感。<a href="https://cloudflare.jove.com/files/ftp_upload/61760/Table_1.xlsx" target="_blank">请单击此处下载此表。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61760/61760fig03.jpg"> 图3:对NM日冕中代谢物的有针对性的分析。吸附到 SiO2和 TiO2 NMs 的 cation。 Red 是指代谢物的初始浓度，而蓝色是指在 37 °C 下与 NM 一起孵育 1 小时后代谢物的浓度。溶液中代谢物浓度的降低是代谢物吸附到 NM 表面的结果。聚苯乙烯 NMs 没有显著吸附代谢物。盒图表示最小值和最大值、中值和四分位数范围。图转载自CESI-MS定向分析下公布的代谢物日冕开放访问知识共享CC BY许可证15。<a href="https://www.jove.com/files/ftp_upload/61760/61760fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61760/61760fig04.jpg"> 图4:对NM日冕中电离子代谢物的有针对性分析，重点是异位体吸附。将离子吸附到一系列泰坦尼亚 NMs 中，显示出各种异位体（如磷酸糖）之间的明显差异。红色是指代谢物的初始浓度，而蓝色是指在 37 °C 下与 NM 孵育 1 小时后代谢物的浓度。溶液中代谢物浓度的降低是代谢物吸附到 NM 表面的结果。SiO2和聚苯乙烯 NMs 没有显著吸附代谢物。 框图表示最小值和最大值、中位数和四分位数范围。图转载自CESI-MS定向分析下出版的代谢物日冕开放访问创意共同CC BY许可证15。<a href="https://www.jove.com/files/ftp_upload/61760/61760fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>分析</td>
			<td>平均 RSD 峰值区域</td>
			<td>平均 RSD 迁移时间</td>
		</tr>
<tr>
<td>1928 年多肽盘中 （n=3）</td>
			<td>15%</td>
			<td>0.30%</td>
		</tr>
<tr>
<td>27 日内 （n=16）</td>
			<td>5.80%</td>
			<td>2.20%</td>
		</tr>
<tr>
<td>27 cation 日间 （n=36）</td>
			<td>8.60%</td>
			<td>1.80%</td>
		</tr>
</tbody></table>表2:多肽和代谢物CESI-MS技术复制的可重复性。RSD:相对标准偏差，作为可重复性的衡量标准。CESI-MS在高峰地区和迁移时间方面的可重复性非常好，所有分析均为RSD<15%，迁移时间<2.2%。这些结果表明，使用这种技术收集的数据具有高度的信心，并确认检测到的变异是由于样本成分（NMs 上的丰富）的真正差异，而不是分析变异。表2是从之前提出的11，15的结果生成的。

Watch this Scientific Journal Video about Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials at JoVE.com

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

在这里，我们提出了一个协议，以描述完整的生物分子日冕，蛋白质和代谢物，从生物流体的纳米材料使用毛细血管电泳 - 质谱法获得。

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 ...

capillary-electrophoresis-mass-spectrometry-approaches-for

Research

JoVE Journal

Chemistry

4.4K 次观看.  University of Birmingham. 在这里，我们提出了一个协议，以描述完整的生物分子日冕，蛋白质和代谢物，从生物流体的纳米材料使用毛细血管电泳 - 质谱法获得。

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

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

科研

生物化学

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

毛细管电泳质谱法提取培养细胞中的水代谢物进行代谢组学分析

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

癌症研究

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 ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

利用毛细管区电泳串联质谱技术进行大尺度自上而下蛋白质组学

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

化学

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

基于毛细管电泳的氢/氘交换，用于自上而下的质谱法对蛋白质进行构象表征

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

大蛋白复合物结构质谱分析

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

生物学

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T波离子迁移质谱蛋白复合物分析的基本实验程序

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

分析甲基化和其他 S腺苷 L</sub捕捉复合质谱（CCMS）>同型半胱氨酸结合蛋白

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

发现蛋白质相互作用和检定蛋白质功能使用的HaloTag技术

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

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

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