Name: a hydrogen-deuterium exchange mass spectrometry (hdx-ms) platform for investigating peptide biosynthetic enzymes
Uploaded: 2020-05-04T13:00:00
Description: Watch this Scientific Journal Video about A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes at JoVE.com

这项工作得到了加拿大自然科学和工程研究理事会、魁北克自然与技术基金会、加拿大创新基金会和麦吉尔大学创业基金的支持。

1. 准备脱毒试剂和酶库存解决方案
<ol><li>将 HDX 反应所需的试剂（包括任何缓冲器、盐、基板、配体等）准备为 D2O 中的 100 −200x 浓缩库存解决方案（99.9% 原子分数 D）。准备至少 50 mL 的缓冲库存解决方案。 注：对于HalM2的描述， 准备了以下解决方案： 500 mM MgCl2，100 mM 三 （2- 卡盒乙基） 磷酸 （TCEP）， 750 mM ATP （在 HEPES 缓冲区）， 800 m HEPES pD 7.1， 500μM 哈拉2 和 500 m M AMPPNP....

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本协议中介绍的 HDX-MS 工作流程为绘制蛋白质结构动态元素的空间分布图和调查这些动力学如何因扰动（脂结合、酶突变等）而变化提供了非常强大的平台。HDX-MS 与其他通常用于研究构象动力学的结构生物学方法相比具有几个明显的优势。最值得注意的是，只需要少量的蛋白质。使用此处描述的工作流程，1 mL 1 μM 蛋白质样本为包含 5 个交换时间点的三方 HDX 反应提供了足够的材料。此外，兴趣蛋...

蛋白质是结构动力学分子，在时间尺度上采样不同的构象，从女性到第二级的键振动到整个蛋白质域的重新排列，这些排列可以在多秒1内发生。这些构象波动通常是酶/蛋白质功能的关键方面。例如，由配体结合引起的构象变化通常对于调节酶功能至关重要，无论是组织催化所需的活动站点残留物、在顺序动能机制中定义基板结合位点、保护反应中间体免受环境影响，还是通过全石器网络调节酶功能。最近的研究还表明，在整个进化过程中可以保存符合性动力学，而对保存分子运动的扰动可以与基质特异性的变化和新酶功能2、3的出现相关。
近年来，氢-铀交换质谱仪（HDX-MS）迅速成为探索蛋白质构象景观如何对配体结合或突变4、5、6、7等扰动做出反应的有力技术。在典型的HDX-MS实验（图1）中，一种感兴趣的蛋白质被放入D2O制备的缓冲器中，这触发了可溶剂交换质子与 deuteria 的替代。肽债券的交换率在很大程度上取决于pH度、局部氨基酸序列和8号辣椒的局部结构环境。从事氢粘结相互作用的中位（如存在于α螺旋和β片中）的交换速度比暴露于散装溶剂的蛋白质非结构化区域中的中位要慢。因此，铀吸收的程度反映了酶的结构。符合动态的酶，或在配体结合后进行结构转换的酶，将有望产生可测量的HDX响应。
结构化中间体缓慢汇率的机械基础见图25、8、9。为了进行HDX，结构区域必须首先对展开的构象进行瞬时采样，以便通过特定的酸/碱基化学机制促进HDX交换的溶剂分子能够进入可交换的中间体。最终，化学汇率（k化学）的相对幅度和折叠和重折叠率（k打开和k关闭）决定了实验5、8中测量的HDX汇率。从这个简单的动能模型中，很明显，铀吸收的程度将反映潜在的构象动力学（如k开和k关闭的定义）。大多数HDX-MS实验都是在自下而上的工作流程中进行的，在交流反应之后，兴趣蛋白被消化成肽，每个肽的吸乳量被测量为质量7的增加。通过这种方式，HDX-MS 允许在肽的局部空间尺度上映射酶构象动力学的扰动，使研究人员能够评估扰动如何改变感兴趣的酶不同区域的动力学。
HDX-MS方法用于阐明蛋白质结构动力学的优点是多种多方面的。首先，该方法可以在具有第四纪结构10的系统中用少量的原生蛋白质或蛋白质复合物进行。只要自下而上的HDX-MS工作流程提供足够数量的自信识别肽，涵盖感兴趣的蛋白质序列，就根本不需要在检测中使用的酶制剂进行高度纯化。此外，HDX-MS可以提供近原生条件下的构象动力学信息，而无需像单分子荧光研究13中那样使用部位特异性蛋白质标签，并且对可以研究的蛋白质或蛋白质复合物没有大小限制（这使得核磁共振[NMR]光谱学等方法具有挑战性）7，14。最后，可以采用时间解决的HDX-MS方法来研究内在紊乱的蛋白质，这些蛋白质很难用X射线晶体学15、16、17、18来研究。HDX-MS 的主要限制是数据结构分辨率低。HDX-MS 数据可用于指出构象动力学的变化位置，并揭示耦合的构象变化，但它们通常不会提供对驱动观测变化的精确分子机制的深入了解。电子捕获分离方法与蛋白质HDX-MS数据相结合的最新进展表明，将交换点绘制为单一氨基酸残留物19有希望，但还需要进行后续生化和结构研究，以便为HDX-MS数据转发的结构模型提供清晰度。
下面，提出了一个详细的协议，为开发HDX-MS检测是20。下面介绍的样品制备协议通常应适用于在水性缓冲中表现出良好溶解性的任何蛋白质。更专业的样品制备方法和HDX-MS工作流程可用于蛋白质比需要检测在洗涤剂或磷脂的存在21，22，23，24。HDX-MS 数据收集的仪器设置被描述为高分辨率四倍飞行时间质谱仪与液相色谱系统相结合。可以在一些市售的液相色谱-质谱 （LC-MS） 系统中的任何一个系统上收集具有类似复杂性和分辨率的数据。还提供了使用市售软件包进行数据处理的关键方面。此外，我们亦提供与更广泛的HDX-MS社区12的建议相一致的数据收集及分析指引。所述协议用于研究HalM2的动态结构特性，HalM2是一种促进抗菌肽天然产品20的多步成熟。我们说明了如何使用 HDX-MS 来揭示基板绑定站点和未采用以前特征的异体特性。近年来，关于蛋白质HDX-MS的其他几项协议已经发表25，26。结合目前的工作，这些早期的贡献应该为读者提供一些实验设计的灵活性。

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>[glu-1]-fibrinopeptide B (Glu-Fib)</td>
			<td>BioBasic</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL Amicon Ultracel 10k centrifugal filtration device (Millipore)</td>
			<td>Milipore Sigma</td>
			<td>UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Fisher</td>
			<td>A955-1</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP-PNP</td>
			<td>SIGMA</td>
			<td>A2647-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>SIGMA</td>
			<td>a2383-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>D2O</td>
			<td>ALDRICH</td>
			<td>435767-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid</td>
			<td>Thermo Fisher</td>
			<td>28905</td>
			<td></td>
		</tr>
		<tr>
			<td>guanidine-HCl</td>
			<td>VWR</td>
			<td>97063-764</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>SiGMA-Aldrich</td>
			<td>63068-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>BioBasic</td>
			<td>PB0440</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium phosphate</td>
			<td>BioBasic</td>
			<td>PB0445</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP Hydrochloride</td>
			<td>TRC Canada</td>
			<td>T012500</td>
			<td>peptide was synthesized upon request</td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deuteros</td>
			<td>Andy M C Lau, et al</td>
			<td>version 1.08</td>
			<td></td>
		</tr>
		<tr>
			<td>DynamX</td>
			<td>Waters</td>
			<td>version 3.0</td>
			<td></td>
		</tr>
		<tr>
			<td>MassLynx</td>
			<td>Waters</td>
			<td>version 4.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Lynx 
			Global Server (PLGS)</td>
			<td>Waters</td>
			<td>version 3.0.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PyMOL</td>
			<td>Schr&#246;dinger</td>
			<td>version 2.2.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Instrument and equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC BEH C18 analytical Column</td>
			<td>Waters</td>
			<td>186002346</td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC BEH C8 VanGuard Pre-column</td>
			<td>Waters</td>
			<td>186003978</td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC M-Class HDX System</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HDX Manager</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microtip pH electrode</td>
			<td>Thermo Fisher</td>
			<td>13-620-291</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Enzymate BEH column or Pepsin solumn</td>
			<td>Waters</td>
			<td>186007233</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Synapt G2-Si</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a hydrogen-deuterium exchange mass spectrometry (hdx-ms) platform for investigating peptide biosynthetic enzymes

氢-铀交换质谱仪（HDX-MS）是酶构象变化和酶基质相互作用生物物理特征的有力方法。HDX-MS 的诸多优点中，仅消耗少量材料，无需酶/基质标签即可在近原生条件下进行，并且可以提供酶构象动力学空间解决信息−甚至用于大型酶和多蛋白复合物。该方法由将兴趣酶稀释到 D2O 中准备的缓冲器中启动。这触发了肽键（N-H）与铀（N-D）的蛋白交换。在所需的交换时间点，反应液被淬火，酶被蛋白酶分解成肽，肽通过超性能液相色谱 （UPLC） 分离，并且每个肽的质量变化（由于氢气交换为铀）由 MS 记录。每种肽吸收的钚量在很大程度上取决于该肽的局部氢结合环境。肽存在于酶交换的非常活跃的区域非常快，而从有序区域提取的肽的交换速度要慢得多。通过这种方式，HDX 速率报告了局部酶构象动力学。然后，在不同配体存在的情况下，对脱脂吸收水平进行扰动，可用于绘制配体绑定位点的地图，识别同位素网络，并了解构象动力学在酶功能中的作用。在这里，我们说明了我们如何使用HDX-MS来更好地了解一种叫做兰西肽的肽天然产品的生物合成。Lanthipeptides 是基因编码肽，通过大型、多功能、符合性动态酶进行转化后进行修饰，这些酶很难用传统的结构生物学方法进行研究。HDX-MS 为研究这些类型的酶的机械特性提供了一个理想且适应性的平台。

有必要评估蛋白催化消化的质量以及每组样品注射工作流程的可重复性。因此，在进行HDX-MS检测之前，必须为兴趣蛋白的蛋白分析、使用反相液相色谱和气体相离子流动性分离肽以及使用MS检测肽建立有效条件。为此目的，应首先调查感兴趣的蛋白质的参考样本（在没有贫血的情况下收集）（第3节）。图3A−C中的色谱数据显示了HalM2兰西肽合成酶三个参考样本的...

Watch this Scientific Journal Video about A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes at JoVE.com

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

在肽天然产品的生物合成过程中，兰西肽合成器催化多步反应。在这里，我们描述了一个连续的，自下而上的，氢-钚交换质谱仪（HDX-MS）的工作流程，可用于研究兰西肽合成器的构象动力学，以及肽天然产品生物合成中涉及的其他类似酶。

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 ...

Research

JoVE Journal

Biochemistry

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

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

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. ...

科研

生物化学

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 ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

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 ...

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 ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

使用自动工作站进行质谱测量的等离子体样品制备

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

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 ...