Name: time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry for studying protein structure and dynamics
Uploaded: 2017-04-17T12:30:00
Description: Watch this Scientific Journal Video about Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics at JoVE.com

We gratefully acknowledge Dr. Markus Zweckstetter for providing the pdb coordinate file for the 'native' tau ensemble predicted from his NMR work, with contributed analysis tools provided by Dr. Adnan Sljoka. Funding for this work was provided by the Natural Science and Engineering Research Council of Canada (NSERC) ENGAGE Grant program.

注意：使用前请咨询所有相关材料安全数据表（MSDS）。由聚（甲基丙烯酸甲酯）的激光烧蚀（PMMA）中产生的烟雾是有毒的。可以肯定的是，激光雕刻机连接到工作通风系统。当构建微流体装置包括利用工程控制（通风柜，锐器容器）和个人防护设备（安全眼镜，面罩，手套，实验室外套，全长长裤，不露脚趾的鞋子），利用一切适当安全措施。这是最重要的是使用高效液相色谱法（HPLC）级试剂?...

<ol>
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虽然结构生物学方法如X射线晶体学和NMR是有利的，因为它们提供的蛋白质的非常详细的结构，这些图片通常是静态的。过渡形式和弱结构化结构域的表征通过继续这些常规方法进行研究时是难以捉摸的。因此，为了获得对这些类型的系统动态的见解是很重要的，在快速的时间尺度上工作。我们已经成功地应用于TRESI-HDX-MS获得关于本地化肽水平充分研究的IDP内发生的构象变化的详细见解。一到这种...

在过去的几十年中，显著的进步已经在设计用来测量蛋白质结构和动态1，2，3，4分析技术的发展做出了。虽然X射线晶体保持用于确定蛋白质结构原理工具，需要高浓度的蛋白质和广泛的优化是必需的，以产生衍射级晶体。蛋白质是难以结晶，如膜结合和本质无序蛋白已经经典研究了氢-氘交换（HDX）NMR 5。但是，在近数十年来，电喷雾电离质谱（ESI-MS）来HDX的耦合已经迅速得到普及6,7。 质谱提供了一个解决方案许多的通过X射线晶体学和NMR所造成的限制。特别地，MS是高度敏感的（nM至μM所需的浓度），而且事实上对蛋白质大小没有限制。另外，MS分析的高占空比允许用于研究蛋白质作为它们经历酶促周转，错误折叠，络合和其它生物相关的过程的可能性。这些过程通常发生在毫秒到秒的时间尺度，并且需要在分析前试剂的快速混合。 时间分辨电喷雾电离（TRESI）Wilson和Konermann在2003年获准反应发展到由ESI-MS伪实时监控。其设置方式并入的毛细管混合器具有连续可调的反应室体积8。该装置由两个同心毛细管，用密封的内毛细管并切成其侧面的凹口，以允许窄间毛细内的混合从凹口与内毛细管（通常2mm）的的端部Y空间。当施加到HDX实验，内毛细血管携带感兴趣的蛋白质，外毛细管携带标记D 2 O中的溶液，然后经历与蛋白质进入可调反应室允许HDX标记前之前直接转印混入ESI资源。 简言之，将HDX依赖于主链酰胺氢发生在溶液中与图9中 ，10个氘原子交换。交换是碱催化的在生理pH下，用酸催化成为在低于pH约260普遍。交换速率基于四个主要因素：pH，温度，溶剂可及性和分子内氢键。作为前两个因素在整个实验中，交换的速率，特别是在肽骨架的酰胺位置保持恒定，主要是dependen吨蛋白质结构11。紧密折叠在α螺旋和β片层广泛，稳定的氢键键合网络的区域将占用在氘大大缓慢的速率相比，循环和无序区（有时不是在所有）12。这允许全球蛋白质分析，其中在结构的扰动（ 例如 ，在聚合或底物结合）导致不同的氘摄取（ 图1）。 动力学毛细管混合器可掺入含有用于氘摄取的定位的蛋白水解室中的微流体平台。此蛋白水解室在低pH，以便有效地淬灭交换反应保持，并且需要固定的酸性蛋白酶中以消化蛋白质为集中式的肽（ 图2）。在毫秒到秒的时间尺度监测骨干交换对于特别重要的内难以构象变化表征为了表征环区，熔融球，和本质无序蛋白质（流离失所）13，14。可替代地，TRESI-HDX也可以被用于表征当前不具有通过X射线晶体学和NMR的方法来解决的原子结构的蛋白质，使用耦合到COREX算法（DX-COREX）方法15,16氘交换。此详细的协议将适用TRESI-HDX研究牛头，境内流离失所者，在这两个它的原始形式，以及它的致病性过度磷酸化状态。虽然原生头是最充分研究国内流离失所者之一，很少有人知道它的淀粉样蛋白生成对应部13。

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:244px;">Poly(methyl methacrylate) or PMMA</td>
			<td style="width:136px;">Professional Plastics</td>
			<td style="width:145px;">SACR.250CCP</td>
			<td style="width:159px;">8.9 cm x 3.8 cm x 0.6 cm</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:244px;">Fused Silica Glass Capillary</td>
			<td style="width:136px;">Polymicro Technologies</td>
			<td style="width:145px;">106815-0018</td>
			<td style="width:159px;">ID: 75&#181;m, OD: 150&#181;m</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">Metal Capillaries</td>
			<td style="width:136px;">McMaster-Carr</td>
			<td style="width:145px;">28 ga &#8211; 89875K97 
			30 ga&#160; - 89875K99</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:244px;">Fluorinated Ethylene Propylene (FEP) Tubing</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">1477 
			1548</td>
			<td style="width:159px;">ID: 0.007&#8221;, OD: 1/16&#8221; 
			ID: 0.020&#8221;, OD: 1/16&#8221;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:244px;">Standard Polymer Tubing Cutter</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">A-327</td>
			<td style="width:159px;">for 1/16&#8221; and 1/8&#8221; OD tubing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Micro Static Mixing Tee</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">M-540</td>
			<td style="width:159px;">for 1/16&#8221; OD tubing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">or</td>
			<td style="width:136px;"></td>
			<td style="width:145px;"></td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:244px;">Stainless Steel Tee, 0.25mm Bore</td>
			<td style="width:136px;">Valco Instruments Co., Inc. (VICI)</td>
			<td style="width:145px;">ZT1C</td>
			<td style="width:159px;">for 1/16&#8221; OD tubing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">PEEK Tee for 1/16&#8221; OD Tubing</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">P-727</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">10-32 Female to Female Luer</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">P-659</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">10-32 PEEK Double-Winged Nut</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">F-300</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Ferrule for 1/16&#8221; OD Tubing</td>
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			<td height="20" style="height:20px;width:244px;">100 Series Rotary Tool</td>
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			<td style="width:145px;">F013010001</td>
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			<td height="20" style="height:20px;width:244px;">Cut-Off Discs</td>
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			<td style="width:159px;">1/64&#8221; thickness</td>
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		<tr height="34">
			<td height="34" style="height:34px;width:244px;">Stereomaster Digital Zoom Microscope</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:145px;">12-563-411</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Soldering Iron</td>
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			<td style="width:145px;">58-6301-2</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">VersaLaser</td>
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			<td height="20" style="height:20px;width:244px;">Syringes</td>
			<td style="width:136px;">Hamilton</td>
			<td style="width:145px;">81220</td>
			<td style="width:159px;">500&#181;L capacity</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Syringe Pumps</td>
			<td style="width:136px;">Harvard Apparatus</td>
			<td style="width:145px;">70-4501</td>
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			<td height="34" style="height:34px;width:244px;">Pepsin from Porcine Gastric Mucosa</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Deuterium Oxide</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:145px;">151882-10X0.6ML</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Acetic Acid</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:145px;">695092-100ML</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">HPLC Grade Water</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Ammonium Acetate</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:145px;">A7330-500G</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Phosphate</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:145px;">S369-500</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Sodium Chloride</td>
			<td style="width:136px;">Fisher Scientific</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">CorelDraw X3</td>
			<td style="width:136px;">Corel</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Molecular Weight Calculator</td>
			<td style="width:136px;"></td>
			<td style="width:145px;">Version 6.49</td>
			<td style="width:159px;">Open Source MS Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">mMass</td>
			<td style="width:136px;"></td>
			<td style="width:145px;">Version 5.5.0</td>
			<td style="width:159px;">Open Source MS Tool</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:244px;">ExPASy FindPept</td>
			<td style="width:136px;">Swiss Institute of Bioinformatics</td>
			<td style="width:145px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">SigmaPlot</td>
			<td style="width:136px;">Systat Software</td>
			<td style="width:145px;">Version 11.0</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">PyMOL</td>
			<td style="width:136px;">Schr&#246;dinger</td>
			<td style="width:145px;">Version 1.5.0.4</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:244px;">Name</td>
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		<tr height="34">
			<td height="34" style="height:34px;width:244px;">QStar Elite Hybrid Q-TOF Mass Spectrometer</td>
			<td style="width:136px;">AB SCIEX</td>
			<td style="width:145px;"></td>
			<td style="width:159px;"></td>
		</tr>
	</tbody>
</table>

time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry for studying protein structure and dynamics

本质上无序蛋白（流离失所）长期以来一直到结构生物学家一个挑战，因为它们缺乏稳定的二级结构元件。氢氘在快速时间标度测量的交换（HDX）是唯一适合于检测简要地填充的结构和氢键网络，允许在天然合奏瞬态构象异构体的表征。 HDX的耦合质谱具有以下几个主要优点，包括高灵敏度，低样本消耗和蛋白质大小没有限制。这种技术在过去的几十年中极大地推进，包括监测毫秒时间尺度HDX标签时间的能力。此外，通过将工作流程HDX到微流体平台容纳的酸性蛋白酶微反应器，我们可以在肽水平本地化动态属性。在这项研究中，时间分辨电喷雾电离质谱（TRESI-MS）耦合到HDX WA小号用于提供残基结构中的tau蛋白的详细图像，以及取决于过度诱发构象变化。

天然和磷酸-τ的消化谱是相似的，分别得到77.1和71.7％的序列覆盖。每种肽的氘摄取值是通过使用内部开发的软件FORTRAN与拟合产生的理论分布所观察到的同位素分布来确定。最佳拟合分布与相关联的氘摄取值一起示出（ 图3a）。摄取动力学曲线，然后产生了，而且通过单指数表达式来描述。在3个或更多的标记倍观察到的所有的肽动力学曲线（N = 3）进行了分?...

Watch this Scientific Journal Video about Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics at JoVE.com

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

构象的灵活性在蛋白质功能的关键作用。在此，我们描述了使用耦合到氢 - 氘交换用于探测驱动功能在有序和无序的蛋白质的快速结构变化的时间分辨电喷雾电离质谱法。

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Research

JoVE Journal

Biochemistry

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

科研

生物化学

Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web

研究与Bio3D-web的蛋白质序列 - 结构 - 动力学关系

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

多肽基基质辅助激光解吸/电离质谱成像中福尔马林固定样品的优化制备工作流

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

用氢-氘交换质谱法定义 Hsp33's 氧化还原调节陪护活动和 Hsp33 映射构象变化

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

叶喷雾质谱: 一种快速的环境电离技术直接评价植物组织中的代谢产物

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

探头电喷电镀质谱的样品制备

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Quaternary Structure Modeling Through Chemical Cross-Linking Mass Spectrometry: Extending TX-MS Jupyter Reports

通过化学交联质谱法进行四元结构建模：扩展 TX-MS Jupyter 报告

Protein-protein interactions can be challenging to study yet provide insights into how biological systems function. Targeted cross-linking mass ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

氢-铀交换质谱仪（HDX-MS）平台，用于研究肽生物合成酶

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...