毫秒氢/氘交换质谱法用于生理条件下α-突触核蛋白结构动力学的研究
Summary
单体α-突触核蛋白的结构集合影响其生理功能和理化性质。本方案描述了如何进行毫秒氢/氘交换质谱和随后的数据分析，以确定在生理条件下这种内在无序蛋白质的单体的构象信息。
Abstract
α-突触核蛋白（aSyn）是一种内在无序的蛋白质，其纤维聚集体在路易体和神经突起中含量丰富，这是帕金森病的标志。然而，它的大部分生物活性以及它的聚集，都集中在蛋白质的可溶性单体形式上。阐明aSyn生物学和病理生理学的分子机制需要结构高度解析的方法，并且对生物学条件敏感。其原生展开的，亚稳态的结构使单体aSyn难以解决许多结构生物学技术。这里描述了一种这种方法的应用：氢/氘交换质谱（HDX-MS）在毫秒时间尺度上用于研究具有低热力学稳定性和保护因子的蛋白质，例如aSyn。在毫秒时间尺度上，HDX-MS数据包含有关aSyn的溶剂可及性和氢键结构的信息，这些信息在较长的标记时间下丢失，最终产生高达氨基酸水平的结构分辨率。因此，HDX-MS可以以高结构和时间分辨率提供有关构象动力学和热力学，分子内和分子间相互作用以及突变或改变对环境条件的结构影响的信息。虽然广泛适用，但演示了如何在单体aSyn中获取，分析和解释毫秒级HDX-MS测量值。
Introduction
帕金森病（PD）是一种神经退行性疾病，影响全球数百万人1。其特征在于在大脑的黑质紧凑区域形成称为路易体和路易神经突的细胞质内含物。已发现这些细胞质包涵体含有内在无序蛋白 aSyn2 的聚集体。在PD和其他突触核蛋白病中，aSyn从可溶性无序状态转变为不溶性，高度结构化的患病状态。在其天然形式中，单体aSyn采用广泛的构象，通过其N-和C-末端之间的长程静电相互作用以及其C-末端和非淀粉样蛋白β组分（NAC）区域3，4，5，6之间的疏水相互作用而稳定。这些稳定相互作用中的任何破坏，例如突变，翻译后修饰和局部环境的变化，都可能导致单体折叠错误，从而触发聚集过程7。
虽然对aSyn8，9，10，11的低聚和纤维形式存在大量研究，但迫切需要研究蛋白质的单体形式，并更好地了解哪些构象是功能性的（以及如何），哪些容易聚集8，9，10，11.由于本质上是无序的，大小只有14 kDa，并且难以结晶，aSyn单体不适合大多数结构生物技术。然而，一种能够测量单体aSyn构象动力学的技术是毫秒HDX-MS，它最近产生了重要的结构观察结果，否则将具有挑战性或不可能获得12，13，14。毫秒 HDX-MS 通过监测酰胺氢的同位素交换来灵敏地测量蛋白质构象集合的平均值，指示溶剂可及性和特定蛋白质区域在毫秒时间尺度上的氢键网络参与情况。有必要强调HDX-MS的毫秒方面，因为由于其原生展开的元稳定性，aSyn表现出非常快的氢交换动力学，其表现形式远低于传统HDX-MS系统的下限。例如，大多数aSyn分子在细胞内条件下在不到1秒的时间内将氢完全交换为氘。现在有几个实验室已经建立了快速混合仪器;在这种情况下，使用快速混合淬火流仪器的原型，该仪器能够以50 ms的死区时间为50 ms，时间分辨率为1 ms执行HDX-MS。虽然毫秒HDX-MS最近在aSyn的研究中非常重要，但它在更广泛地研究本质无序的蛋白质/区域以及大量具有仅弱稳定的环/区域的蛋白质方面是有价值的。例如，多肽药物（例如，胰岛素;普洛斯-1/胰高血糖素;tirzepatide）和肽融合蛋白（例如，HIV抑制剂FN3-L35-T1144）是主要的药物形式，其中溶液相结构和稳定性信息可以成为药物开发决策的关键输入，然而，肽部分通常只有HDX-MS在秒级16，17，18，19，20的微弱稳定性和难处理.在秒/分钟结构域中标记的紧急HDX-MS方法已被证明可以推导出DNA G-四链体的结构信息，但应该可以通过应用毫秒级HDX-MS21将其扩展到更多样化的寡核苷酸结构。
HDX-MS实验可以在三个不同的水平上进行：（1）自下而上（标记的蛋白质被蛋白水解消化），（2）中下（标记的蛋白质被蛋白水解消化，所得的肽通过软片段化技术进一步片段化），以及（3）自上而下（软片段化技术直接片段化标记的蛋白质）22.因此，亚分子HDX-MS数据使我们能够将交换行为定位到蛋白质的特定区域，因此对于此类实验具有足够的序列覆盖率至关重要。任何HDX-MS实验的结构分辨率分别依赖于消化或软片段化时从蛋白质衍生的蛋白水解肽或片段的数量。在上面概述的三种实验类型中的每一种中，每个肽/片段的酰胺交换的变化被映射回蛋白质的主要结构，以指示蛋白质局部区域的行为。虽然最高的结构分辨率是通过软碎片实现的，但这些实验的描述超出了当前研究的范围，该研究的重点是测量aSyn单体构象。通过此处描述的常用“自下而上”工作流程可以获得出色的结果。
这里提供了以下程序：（1）如何制备和处理aSyn样品和HDX-MS缓冲液，（2）如何为自下而上的HDX-MS实验进行肽图分析，（3）如何在生理条件下，特别是在毫秒时域中获取单体aSyn上的HDX-MS数据（使用定制的仪器;用于毫秒标记的替代仪器） （4）如何处理和分析HDX-MS数据。这里举例说明了在生理pH（7.40）下在两种溶液条件下使用单体aSyn的方法。虽然在aSyn的研究中至关重要，但这些程序可以应用于任何蛋白质，而不限于内在无序的蛋白质。
Protocol
1. 同步蛋白的蛋白表达和纯化 按照以前发布的报告准备 aSyn9. 透析到安全的储存缓冲液中（例如，Tris，pH 7.2）。 如果需要，浓缩样品（例如，使用3 kDa MWCO，14，000 x g 约10-30分钟的旋转过滤微量离心管，参见 材料表）。注意：建议不要过度集中注意力。单体融合的完整性尚未超过25 μM得到验证。 等分试样并储存?…
Representative Results
由于其固有的无序性质，很难捕获aSyn在生理pH下错综复杂的结构变化。HDX-MS监测主链酰胺氢的同位素交换，探测蛋白质构象动力学和相互作用。它是在高结构和时间分辨率下获取此信息的少数技术之一。该协议广泛适用于各种蛋白质和缓冲液条件，例如在两种不同的溶液条件下测量aSyn的交换动力学：状态A和状态B8，如步骤2.1.-2.2中定义。 首先，在aSyn上进行?…
Discussion
在本文中，描述了以下程序：（1）在单体aSyn上进行肽图分析实验以获得最高的序列覆盖率，（2）在生理条件下获取单体asyn的毫秒级HDX-MS数据，以及（3）对所得HDX-MS数据进行数据分析和解释。所提供的程序通常易于执行，每个标记实验通常仅持续约8小时，用于三个重复和八个时间点，并且映射实验仅持续约2小时。鉴于这里使用的全自动仪器，可以在1天内获取完整的数据集。然而，在处理样品?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
NS由大学理事会钻石禧年奖学金资助。JJP由UKRI未来领袖奖学金支持[资助编号：MR / T02223X / 1]。
Materials
| 1 × 100 mm ACQUITY BEH 1.7 μm C18 column | Waters Corporation | 186002346 | Analytical column |
| Acetonitrile HPLC grade >99.9% HiPerSolv | VWR | 20060.420 | For LC mobile phases |
| CaCl2 | Sigma Aldrich | C5670 | Salt for HDX buffers |
| Chronos | Axel Semrau (Purchased from Waters Corporation) | 667006090 | Scheduling software to enable multiple HDX-MS sample injections automatically. Alternative software is available from other vendors e.g. HDXDirector or LEAP Shell |
| Deuterium chloride | Goss Scientific (Cambridge Isotope Laboratories) | DLM-2-50 | For HDX labelling buffers |
| Deuterium oxide (99.9% D2O) | Goss Scientific (Cambridge Isotope Laboratories) | DLM-4 | Deuterated water |
| DynamX 3.0 | Waters Corporation | 176016027 | Isotopic assignment and deuterium incorporation calculation |
| Enzymate BEH Pepsin Column | Waters Corporation | 186007233 | Pepsin digestion column |
| Formic Acid, 99.0% LC/MS Grade | Fisher Scientific | 10596814 | For LC mobile phases |
| Guanidinium hydrochloride | Sigma Aldrich | RDD001-500G | Chaotrope/Denaturant |
| HDfleX | University of Exeter | N/A | https://ore.exeter.ac.uk/repository/handle/10871/127982 |
| KCl | Sigma Aldrich | P3911 | Salt for HDX buffers |
| LEAP HDX-2 CTC PAL sampling robot | Waters Corporation | 725000637 | Autosampler robot |
| Leucine enkephalin | Waters Corporation | 186006013 | For mass spectrometry lockspray calibration. |
| MassLynx | Waters Corporation | 667004007 | Software controlling inlet methods and mass spectrometer |
| Maximum recovery vials | Waters Corporation | 600000670CV | 100 pack including caps – used for quench tray in LEAP HDX-2 |
| MgCl2 | Sigma Aldrich | M8266 | Salt for HDX buffers |
| Millipore 0.22 µm syringe filters | Millipore | N9CA7069B | Syringe filters |
| ms2min | Applied Photophysics Ltd | N/A | fast-mix quench-flow millisecond hdx instrument |
| NaCl | Sigma Aldrich | S9888 | Salt for HDX buffers |
| Peltier temperature controller | LEAP Technologies Inc. | HP115-COOL/D | Peltier controller to set precise temperature of chambers in the LEAP robot. |
| ProteinLynx Global Server 3.0 | Waters Corporation | 715001030 | Peptide identification software. Alternative software is available from other vendors. |
| Reagent pot caps | Waters Corporation | 186004632 | 100 pack |
| Reagent pots for LEAP HDX-2 | Waters Corporation | 186001420 | 100 pack excluding caps – used for buffers in LEAP HDX-2 |
| Sodium deuteroxide (99.5% in D2O) | Goss Scientific (Cambridge Isotope Laboratories) | DLM-57 | For HDX labelling buffers |
| Spin filter microcentrifuge tubes (3 kDa MWCO) | Amicon (Merck Sigma Aldrich) | UFC5003 | Micro centrifuge tubes to concentrate protein. This facilitates buffer exchange and accurate sample loading for HDX-MS experiments. |
| Synapt G2-Si mass spectrometer | Waters Corporation | 176850035 | Mass spectrometer |
| Total recovery vials | Waters Corporation | 600000671CV | 100 pack including caps – used for labelling tray in LEAP HDX-2 |
| Tris-HCl | Sigma Aldrich | T3253-250G | Buffer |
| Trizma base | Sigma Aldrich | T60040-B2005 | Buffer |
| Urea | Sigma Aldrich | U5378-1KG | Chaotrope/Denaturant |
| VanGuard 2.1 x 5 mm ACQUITY BEH C18 column | Waters Corporation | 186004623 | Trap desalting column |
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Diesen Artikel zitieren
Seetaloo, N., Phillips, J. J. Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions. J. Vis. Exp. (184), e64050, doi:10.3791/64050 (2022).