Name: capillary electrophoresis-based hydrogen/deuterium exchange for conformational characterization of proteins with top-down mass spectrometry
Uploaded: 2021-06-08T14:00:00
Description: Watch this Scientific Journal Video about Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry at JoVE.com

这项工作得到了国家自然科学基金（NSFC 21974069）的资助。作者还得到了中国深圳湾实验室细胞分析研究所的支持。江苏省生物医用功能材料协同创新中心;南京师范大学江苏省生物医用材料重点实验室。

注意：尽可能使用高效液相色谱（HPLC）级或MS级试剂，以最大限度地减少可能干扰MS分析的污染物。测量过程中不要徒手触摸CE-MS接口，以免因电泳电压或电喷雾电压而造成电击。
1. 材料准备
<ol><li>熔融石英毛细管改性用于CE<ol>
<li>通过将HPC粉末（分子量[MW]：100 kDa）溶解在水中并在室温下在磁力搅拌器上连续搅拌约12小时或直到固体颗粒12完全消失，制备5%?...

<ol>
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涂覆CE毛细管内壁的目标包括在CE过程中最小化电渗流和蛋白质吸收13。虽然电渗流有利于传统的小分子CE分析，因为它能够将中性或带相反电荷的物质驱动到检测器，但它会损害溶液中具有相似尺寸和净电荷的蛋白质物种的分离效率。用HPC涂覆毛细管可最大限度地减少由毛细管内壁上的硅烷醇基团引起的电渗流。此外，掩盖这些硅醇基团减少了它们与蛋白质的相互作用，避免了?...

区分不同构象、结合或修饰状态的蛋白质物种并表征其结构差异对于监测这些参与生物事件的物种之间的转变途径非常重要，从分子识别到酶催化，并了解这些事件背后的机制。传统的生物物理技术由于分辨率不足和溶液中动态信息丢失等局限性而无法提供完整的解决方案。氢/氘交换与质谱（HDX MS）相结合是一种技术，通过蛋白质不稳定的氢原子之间的交换和故意引入的2 H 2O溶液中的2H之间的交换，用氘（2H）标记蛋白质的结构和构象特征。参与氢键或从蛋白质内部的溶剂中螯合的质子不易交换1.因此，由于可交换位点的交换速率高度依赖于其参与高阶结构，因此可以通过MS以高空间分辨率揭示蛋白质结构，该MS基于1H和2H之间的不同原子质量探测2H摄取的范围和速率。近几十年来，HDX MS已成为研究蛋白质构象和动力学的一种非常成功的技术2。
在HDX MS的经典自下而上方法中，不同构象，结合或修饰状态的蛋白质物种的集合在完整蛋白质水平上不分离的情况下进行蛋白水解，因此通过分析具有卷曲氘含量的所得蛋白水解片段来表征单个物种是不可行的。相反，在自上而下的方法中，不同的蛋白质状态或蛋白形式在MS扫描中掺入了不同的氘含量，从而产生完整蛋白质团块的多个分布。这允许通过使用适当的质量过滤器（例如四极杆）的质量选择对应于每个质量分布的离子的质量选择来分离单个物种，并在随后的串联MS分析3，4，5，6中表征其构象差异。然而，在该策略中分离蛋白质状态或蛋白形式的有效性受到其相应质量分布差异程度的限制。
毛细管电泳（CE）提供了一种基于其在溶液相中的不同电荷和流体动力学尺寸以高效率分离蛋白质种类的方法7。将CE与HDX相结合，可以在溶液相中额外分离蛋白质状态或蛋白形式。此外，CE毛细管体积小，允许利用完全氘代溶液作为背景电解质溶液（BGE），即电泳缓冲液，使毛细管成为蛋白质样品的HDX反应器。由于在电泳过程中蛋白质和质子试剂之间的电泳迁移率存在差异，在CE期间进行HDX会导致蛋白质分析物的几乎完全氘化环境，残留的非氘代含量最小，从而提高使用HDX数据进行结构分析的灵敏度。因此，我们开发了一种基于CE的差分HDX方法，结合自上而下的MS，以状态或蛋白形式特异性方式表征蛋白质高阶结构8。
本文通过详细说明材料制备，实验程序和数据分析的步骤来描述这种方法的方案。可能影响方法性能或数据质量的因素在简短的注释中列出。这里介绍的代表性结果包括不同蛋白质混合物的差异HDX数据和牛β-乳球蛋白（β-lg）的天然变体，牛奶中存在的主要乳清蛋白9。我们证明了β-lg的两个丰富变体（即A和B10，11）在基于CE的HDX期间的分离效率，再现性和2H标记性能，以及它们构象的变体特异性表征。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ammonium acetate</td>
			<td>Fisher Chemical</td>
			<td>A/3446/50</td>
			<td>&#8805;99%</td>
		</tr>
		<tr>
			<td>CESI 8000 plus capillary electrophoresis system</td>
			<td>Sciex, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf</td>
			<td>5406000097</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifugal filter</td>
			<td>Merck</td>
			<td>UFC201024</td>
			<td>10 kDa cutoff</td>
		</tr>
		<tr>
			<td>deuterium oxide</td>
			<td>Energy Chemical</td>
			<td>E090001</td>
			<td>99.9 % D</td>
		</tr>
		<tr>
			<td>formic acid</td>
			<td>Acros Organics</td>
			<td>&#160;270480250</td>
			<td></td>
		</tr>
		<tr>
			<td>fused silica glass capillary</td>
			<td>Polymicro Technologies</td>
			<td>1068150017</td>
			<td>ID 50&#956;m, OD 360&#956;m</td>
		</tr>
		<tr>
			<td>gas chromatography</td>
			<td>Agilent</td>
			<td>GC6890N</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrochloric acid</td>
			<td>Sigma Aldrich</td>
			<td>258148</td>
			<td></td>
		</tr>
		<tr>
			<td>hydroxypropyl cellulose</td>
			<td>Aladdin</td>
			<td>H113415</td>
			<td>MW 100000</td>
		</tr>
		<tr>
			<td>magnetic stirrers</td>
			<td>DLAB</td>
			<td>8030101212</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Fisher Chemical</td>
			<td>A456-4</td>
			<td>MS grade</td>
		</tr>
		<tr>
			<td>microvolume UV-Vis spectrophotometer</td>
			<td>DeNovix</td>
			<td>84677JK7731</td>
			<td></td>
		</tr>
		<tr>
			<td>myoglobin</td>
			<td>Sigma Aldrich</td>
			<td>M1882</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbitrap Fusion Lumos mass spectrometer</td>
			<td colspan="2">Thermo Fisher Scientific, USA</td>
			<td></td>
		</tr>
		<tr>
			<td>PA 800 Plus Pharmaceutical Analysis CE System</td>
			<td>Beckman Coulter, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Q Exactive UHMR mass Spectrometer</td>
			<td>Thermo Fisher Scientific, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium&#160;hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>ubiquitin</td>
			<td>Sigma Aldrich</td>
			<td>U6253</td>
			<td></td>
		</tr>
		<tr>
			<td>ultrasonicator</td>
			<td>SCIENTZ</td>
			<td>SB-5200</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-lactoglobulin</td>
			<td>Sigma Aldrich</td>
			<td>L0130</td>
			<td></td>
		</tr>
	</tbody>
</table>

capillary electrophoresis-based hydrogen/deuterium exchange for conformational characterization of proteins with top-down mass spectrometry

解决溶液中共存的多种蛋白质状态的构象非均质性仍然是蛋白质治疗药物表征和确定对生物学功能至关重要的构象过渡途径（从分子识别到酶催化）的主要障碍之一。氢/氘交换（HDX）反应与自上而下的质谱（MS）分析相结合，提供了一种以构象特异性方式表征蛋白质高阶结构和动力学的方法。该技术的构象分辨能力高度依赖于在完整蛋白质水平上分离蛋白质状态的效率，并最大限度地减少HDX反应期间残留的非氘代质子含量。 
在这里，我们描述了HDX MS方法的基于毛细管电泳（CE）的变体，旨在提高构象分辨率。在这种方法中，蛋白质在毛细管电泳分离过程中通过氘代背景电解质溶液（BGE）迁移时进行HDX反应。在溶液中共存的不同蛋白质状态或蛋白质形式可以基于其不同的电荷大小比有效地分离。蛋白质和质子溶剂分子之间电泳迁移率的差异最大限度地减少了残留的非氘代溶剂，从而在HDX过程中产生了几乎完全的氘化环境。流通的微粒CE-MS界面允许与喷雾器出口处的淬灭和变性改性剂溶液快速混合后，对洗脱的蛋白质物种进行有效的电喷雾电离。在线自上而下的MS分析测量洗脱完好的完整蛋白质物种的全球氘化水平，随后测量其气相片段的氘化。本文在差分HDX系统中演示了这种方法，包括牛奶中共存的天然蛋白质变体。

改变BGE的输注压力可以调整分离效率和迁移时间，这相当于要分离的蛋白质的HDX反应时间（图3）。较低的输注压力导致以实验持续时间为代价更好地分离CE峰（图3A）。较长的迁移/ HDX反应时间导致蛋白质分析物的更高水平的氘化（图3B-D）。在 HDX 分钟时间尺度上，氘变差异应主要反映结构受保护站点（?...

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

这里介绍的是基于毛细管电泳的氢/氘交换（HDX）方法与自上而下的质谱法相结合的方案。该方法通过同时进行差分HDX和电泳分离来表征不同蛋白质物种（包括不同状态的蛋白质和不同蛋白质形式）之间高阶结构的差异。

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 ...

该方法允许在单个CEMS测量中完成共存蛋白质物种的分离，包括不同的构象或变体，并在线表征其高阶结构差异。它利用电泳效应为HDX反应和HDX期间共存蛋白质物种的正交分离提供近100%持续时间的环境，以进行自上而下的MS分析。从毛细管电泳的预处理，氢氘交换或CE HDX设置开始。在50%甲醇，49%水和1%甲酸的混合物中超声处理，在室温下清洁通过微样品CE-MS界面的流动至少30分钟。将 HPC 改良毛细管安装在毛细管电泳或 CE 仪器上。使用自动进样器用背景电解质溶液或BGE冲洗毛细管10分钟，并使其充满BGE。使用适当长度的未改性二氧化硅毛细管作为改性剂溶液的输注管。使用活接和适当的套管将改性剂管连接到具有钝温度的气密玻璃注射器，并使用输液泵用改性剂溶液冲洗管子至少10分钟。将装有相应溶液的 HPC 编码 CE 毛细管和改性剂管的出口插入清洁的 CE-MS 接口。使用输液泵推进注射器，使改性剂溶液到达接口的尖端。将组装好的CE-MS接口安装在质谱仪的纳米电子喷雾电离源外壳上。向 CE-MS 接口施加 3-5 千伏的喷雾电压。将改性剂溶液与注入器泵一起以每分钟0.1-10微升的流速注入，并确保在CE-MS接口的尖端进行稳定的电喷雾。放置含有BGE和自动采样器的样品瓶，稍后将用于获取空白电泳图和空白质谱。使用注射量和注射参数之间的关系估计注射量。并使用自动进样器以两个PSI进样样品溶液，持续适当的持续时间。通过在0-2 PSI范围内的输注压力下施加20千伏的电泳电压来开始CE分离，并获得电泳图。同时，在色谱模式下获取MS数据，其中离子电流图作为时间的函数获取，并且相应的MS扫描不会自动组合成单个光谱。保存空白电泳图和质谱作为参考。将含有所需浓度蛋白质样品溶液的样品瓶置于自动进样器中，并获取电泳分离和氘标记蛋白质物种的电泳图和MS1光谱。对感兴趣的物种执行串联 Ms.测量，在同一运行中获取 MS1 光谱后，或在随后的单独运行中。每次测量后，用BGE在20 PSI的压力下冲洗毛细管至少10分钟。在实验完成后，清洁CEMS接口和所有管道以进行存储。在直接输注模式下使用 MS 获取 HDX 端点样本的数据集。低输注压力导致CE峰的分离更好，但导致迁移时间和实验持续时间的增加。较长的 HDX 反应时间导致蛋白质分析物的去累水平较高。β免疫球蛋白或β-IG的A和B方差，其序列中仅存在两个氨基酸残基的差异，在EIC衍生的电泳图中产生了两个充分分离的峰。差异的氘标记方差导致离子的质量分布不同。Z离子的相对丰度低于C离子，因为在确认βIG时存在独特的异构连接，这限制了CS 82和CS 176之间的碎片化效率。β IGA产生的大多数片段离子和β IGB表现出相似程度的氘摄取。然而，覆盖β IGA序列变异位点的较大片段的贬值程度明显高于β IGB。BGE的pH值根据样品的等电点进行调整，以防止蛋白质聚集，并在保持复合物的同时促进分离。

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Principles of Mass Spectrometry

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Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

使用氢交换质谱分析蛋白质动力学

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

科研

化学

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

亚毫秒构象变化蛋白的光热光束偏转已解决

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

环境质谱可视化纹影摄影的使用

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

液相色谱离子迁移 QTOF 质谱测定蛋白质生物中宿主细胞蛋白的 HS-MRM 法

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

活蛙 (爪蟾) 胚胎单细胞代谢的微探针毛细管电泳质谱

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

基质辅助激光解吸电离飞行时间 (MALDI) 质谱法表征合成聚合物

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

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

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 ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS) for Quantification of Iron Redox Species (Fe(II), Fe(III))

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry CE-ICP-MS for Quantification of Iron Redox Species FeII, FeIII

为铁氧化还原物种（Fe（II）、Fe（III）定量设置毛细管电泳-电感耦合等离子体质谱仪（CE-ICP-MS）

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative ...

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

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 ...