无激光羟基基蛋白足迹，对蛋白质进行更高级的结构分析
Summary
该协议提出了一种使用内联基量程和等离子光源进行闪光氧化蛋白足迹的方法。该方法取代了危险的紫外激光器，简化和提高了蛋白质研究快速光化学氧化的可重复性。
Abstract
羟基基蛋白足迹 （HRPF） 是一种新兴且很有前途的高阶结构分析技术，可提供有关蛋白质结构、蛋白质-蛋白质相互作用或蛋白质-脂蛋白相互作用变化的信息。HRPF 利用羟基基（▪OH） 不可逆转地标记蛋白质的溶剂可访问表面。执行HRPF固有的复杂性、成本和危险性大大限制了生物制药的广泛采用。这些因素包括：1） 使用复杂、危险和昂贵的激光，需要采取重大安全防范措施：和 2） 由于背景清理▪ OH而导致的 Hrpf 的不可复制性限制了比较研究。本出版物为无激光HRPF系统的运行提供了协议。这种无激光HRPF系统采用高能、高压等离子光源闪光氧化技术，采用内联基量程法。等离子光源比激光型HRPF系统更安全、更易于使用，在产生羟基基方面效率更高，并且在线基多厘米可增加研究的可重复性。无激光HRPF系统结合，解决并克服了上述激光技术的缺点和局限性。
Introduction
蛋白质构象和相关的高阶结构（HOS）是正常生物功能和异常行为的主要决定因素1。这同样适用于生物制药，其结构和功能活动取决于其生产和环境的各个方面。HOS的生物制药变化与不良药理学和患者免疫反应2、3的不良药物反应（ADR）有关。ADR的出现提醒生物制药行业注意蛋白质HOS在生物治疗剂的安全性和有效性中所起的关键作用，并且他们已确定需要新的和改进的HOS分析4。
羟基基蛋白足迹 （HRPF） 是跟踪蛋白质 HOS 变化的一项很有前途的技术。HRPF 涉及蛋白质外部不可逆转的标记，▪OH，然后是质谱 （MS） 分析，以确定蛋白质5、6、7的溶剂可访问表面。HRPF已成功地用于检测蛋白质HOS的缺陷及其功能8，9，特征为单克隆抗体（mAb）10，11，12，13的HOS，确定一个配体14的结合Kd，以及更多的15，16，17，18，19。产生HRPF的▪OH的常用方法是蛋白质的快速光化学氧化（FPOP），它使用高能、快速紫外线激光器从H2O2的光解中产生▪OH。在大多数情况下，FPOP使用昂贵的驱魔激光器使用有害气体（KrF），需要实质性的保障措施，以避免呼吸和眼睛受伤20。为了避免吸入危害，其他人使用频率翻倍的钛钛石榴石（Nd：YAG）激光器21，它消除了有毒气体的使用，但仍然昂贵，需要大量的操作专业知识，并要求广泛的杂散光控制，以保护用户免受眼睛损伤。
虽然可以利用HRPF获得充足的信息，但生物制药的广泛采用尚未得到满足。有限采用HRPF的两个障碍包括：1）使用危险和昂贵的激光，需要大量的安全防范措施20：和 2） 人力资源公积金的不可复制性造成的背景清理 ▪OH 限制比较研究22。为了取代激光的使用，开发了高速、高能量等离子闪光光解装置，以便于安全地执行FPOP。为了提高HRPF实验的不可生产性，实施实时激进量度。
由于背景清理了OH22，HRPF的实践受到不可生产性的限制，这归因于▪OH22的背景清理。虽然▪OH是蛋白质地形的优秀探针，但它们也与制剂中发现的许多成分发生反应，因此有必要测量可用于氧化靶蛋白的基蛋白的有效浓度。缓冲制备、过氧化氢浓度、配体特性或光解的变化可能导致控制组和实验组之间的氧化差异，这些差异在 HOS 差异研究中产生模糊性。增加实时基度量可以调整 OH 负载▪效果，从而在 HRPF 实验中增加置信力和可重复性。在FPOP中使用激进的学分法在其他地方被描述为23，24，25，并在最近出版的26号出版物中进一步详细讨论。在这里，我们描述了使用一种新的闪光光解系统和实时文量法来标记马的apo-肌红蛋白（aMb），比较了FPOP实验中肽氧化水平与使用兴奋激光时获得的水平。
Protocol
1. 安装毛细管 使用硅裂解石，将 250μm 内径 （ID） 二氧化硅毛细丝切成 27 英寸。检查毛细细毛的末端，获得干净、直切的切口。 通过燃烧聚酰胺涂层，创建两个长度约 15 毫米的窗户。从”低端”开始，使第一个光解窗口距离”低端”90毫米，第二个圆点窗口距离”低端”225毫米。注意：一旦涂层被烧毁，毛细细毛是非常脆弱的。 拧开5号端口的螺母和铁杉，在铁杉圆锥端（<strong…
Representative Results
高压等离子体源与实时量表相结合，能够更好地控制▪OH 产量，更准确地观察高阶蛋白质结构的变化。添加腺苷可以有效地实时激进量度。氧化后，腺苷在265纳米（图2A）时失去紫外线吸收性。腺苷吸收的变化与HRPF可用的激进分子的浓度直接相关，从而提供了一种手段，在缓冲器、辅料和配体等激进拾荒者在场的情况下有效监测激进浓度的变化（图2B…
Discussion
在任何HRPF实验中，有几个关键步骤可以确保蛋白质的正确标记。首先，选择适当的流速和源闪速，以确保样品的每个玻色子都照射一次。这确保了蛋白质暴露在OH新形成 的▪的单一波卢斯中。一旦蛋白质被氧化，高阶的蛋白质结构就会改变。要确信原生蛋白质结构被探测到，每个蛋白质分子必须在一瞬间被修改。多西姆可用于测试原生蛋白质结构是否被探测。当羟基基的浓度增加氧化程?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由国家普通医学科学研究所（R43GM125420和R44GM125420）资助。
Materials
| 15 mL Conical Centrifuge Tubes | Fisher Scientific | 14-959-53A | any brand is sufficient |
| 50 µL SGE Gastight Syringes | Fisher Scientific | SG-00723 | |
| Acclaim PepMap 100 C18 nanocolumn (0.75 mm X 150 mm, 2 µm) | Thermo Scientific | ||
| Acetonitrile with 0.1% Formic Acid (v/v), LC/MS Grade | Fisher Scientific | LS120-500 | |
| Apomyoglobin | Sigma-Aldrich | ||
| Catalase | Sigma-Aldrich | C9322 | |
| Centrifuge | Eppendorf | 022625501 | |
| Delicate Task Wipers | Fisher Scientific | 06-666A | |
| Hydrogen Peroxide | Fisher Scientific | H325-100 | any 30% hydrogen peroxide is sufficient |
| Methionine amide | Chem-Impex | 03109 | |
| Microcentrifuge | Thermo Scientific | 75002436 | |
| Orbitrap Fusion Lumos Tribrid Mass Spectrometer | Thermo Scientific | Orbitrap Fusion Lumos Tribrid Mass Spectrometer | other high resolution instruments (e.g. Q exactive Orbitrap or Orbitrap Fusion) can be used |
| Pierce Trypsin Protease, MS Grade | Thermo Scientific | 90058 | |
| Polymicro Cleaving Stone, 1" x 1" x 1/32” | Molex | 1068680064 | any capillary tubing cutter is sufficient |
| UPLC | Thermo Scientific | ||
| Water with 0.1% Formic Acid (v/v), LC/MS Grade | Fisher Scientific | LS118-500 | |
| Water, LC/MS Grade | Fisher Scientific | W6-4 |
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Cite This Article
Weinberger, S. R., Chea, E. E., Sharp, J. S., Misra, S. K. Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins. J. Vis. Exp. (172), e61861, doi:10.3791/61861 (2021).