Name: laser-free hydroxyl radical protein footprinting to perform higher order structural analysis of proteins
Uploaded: 2021-06-04T13:00:00
Description: Watch this Scientific Journal Video about Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins at JoVE.com

这项工作由国家普通医学科学研究所（R43GM125420和R44GM125420）资助。

1. 安装毛细管
<ol><li>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。</li>
	<li>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。</li>
	<li>拧开5号端口的螺母和铁杉，在铁杉圆锥端（<strong...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compensated+Hydroxyl+Radical+Protein+Footprinting+Measures+Buffer+and+Excipient+Effects+on+Conformation+and+Aggregation+in+an+Adalimumab+Biosimilar.">Compensated Hydroxyl Radical Protein Footprinting Measures Buffer and Excipient Effects on Conformation and Aggregation in an Adalimumab Biosimilar.</a> American Association of Pharmaceutical Scientists Journal. 21 (5), 87 (2019).</li><li>Olson, L. J., Misra, S. K., Ishihara, M., Battaile, K. P., Grant, O. C., Sood, A., Woods, R. J., Kim, J. P., Tiemeyer, M., Ren, G., Sharp, J. S., Dahms, N. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enabling+Real-Time+Compensation+in+Fast+Photochemical+Oxidations+of+Proteins+for+the+Determination+of+Protein+Topography+Changes.">Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes.</a> Journal of Visualized Experiments. 163, (2020).</li><li>Hambly, D. M., Gross, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+flash+photolysis+of+hydrogen+peroxide+to+oxidize+protein+solvent-accessible+residues+on+the+microsecond+timescale.">Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale.</a> Journal of American Society of Mass Spectrometry. 16 (12), 2057-2063 (2005).</li><li>Liu, X. R., Zhang, M. M., Gross, M. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+photochemical+oxidation+of+proteins+for+epitope+mapping.">Fast photochemical oxidation of proteins for epitope mapping.</a> Analytical chemistry. 83 (20), 7657-7661 (2011).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+Energetic+Epitope+of+an+Antibody/Interleukin-23+Interaction+with+Hydrogen/Deuterium+Exchange,+Fast+Photochemical+Oxidation+of+Proteins+Mass+Spectrometry,+and+Alanine+Shave+Mutagenesis.">Mapping the Energetic Epitope of an Antibody/Interleukin-23 Interaction with Hydrogen/Deuterium Exchange, Fast Photochemical Oxidation of Proteins Mass Spectrometry, and Alanine Shave Mutagenesis.</a> Analytical chemistry. 89 (4), 2250-2258 (2017).</li><li>Liu, X. R., Zhang, M. M., Rempel, D. L., Gross, M. 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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?...

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。

<table>
	<tbody>
		<tr>
			<td>15 mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-53A</td>
			<td>any brand is sufficient</td>
		</tr>
		<tr>
			<td>50 &#181;L SGE Gastight Syringes</td>
			<td>Fisher Scientific</td>
			<td>SG-00723</td>
			<td></td>
		</tr>
		<tr>
			<td>Acclaim PepMap 100 C18 nanocolumn (0.75 mm X 150 mm, 2 &#181;m)</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile with 0.1% Formic Acid (v/v), LC/MS Grade</td>
			<td>Fisher Scientific</td>
			<td>LS120-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Apomyoglobin</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma-Aldrich</td>
			<td>C9322</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>022625501</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate Task Wipers</td>
			<td>Fisher Scientific</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>Fisher Scientific</td>
			<td>H325-100</td>
			<td>any 30% hydrogen peroxide is sufficient</td>
		</tr>
		<tr>
			<td>Methionine amide</td>
			<td>Chem-Impex</td>
			<td>03109</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002436</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbitrap Fusion Lumos Tribrid Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>Orbitrap Fusion Lumos Tribrid Mass Spectrometer</td>
			<td>other high resolution instruments (e.g. Q exactive Orbitrap or Orbitrap Fusion) can be used</td>
		</tr>
		<tr>
			<td>Pierce Trypsin Protease, MS Grade</td>
			<td>Thermo Scientific</td>
			<td>90058</td>
			<td></td>
		</tr>
		<tr>
			<td>Polymicro Cleaving Stone, 1&#34; x 1&#34; x 1/32&#8221;</td>
			<td>Molex</td>
			<td>1068680064</td>
			<td>any capillary tubing cutter is sufficient</td>
		</tr>
		<tr>
			<td>UPLC</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water with 0.1% Formic Acid (v/v), LC/MS Grade</td>
			<td>Fisher Scientific</td>
			<td>LS118-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, LC/MS Grade</td>
			<td>Fisher Scientific</td>
			<td>W6-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser-free hydroxyl radical protein footprinting to perform higher order structural analysis of proteins

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。

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...

Watch this Scientific Journal Video about Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins at JoVE.com

Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins

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。

无激光羟基基蛋白足迹，对蛋白质进行更高级的结构分析

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes ...

无激光羟基基蛋白足迹使识别蛋白质相互作用点和确认变化区域更加容易，加速了蛋白质聚合、结构和稳定性研究。通过内联基度量程，用户可以实时调整有效的羟基基负荷，使用实时基度文量计，可以节省实验时间和珍贵的样品，同时提高标签的可重复性。首先，用硅裂石将250微米内径的二氧化硅毛细细丝切成27英寸，检查毛细金的末端是否干净而直切。创建两个长度约 15 毫米的窗口。从毛细管的下端，烧掉聚酰胺涂层 90 毫米，以创建光解窗口和 225 毫米的多厘米窗口。插入毛细细纸的下端，正好超过螺母和铁杉的圆锥端。将毛细金加入第五端口，用扳手紧贴手指。通过将其直接拉出，取出光解模块的光解单元盖，然后取出磁性安装的金属面膜，该面膜将保持毛细细线到位。通过按下左边的标签打开圆分细胞，并将圆圆体单元向右摆动。取出磁性安装的夹子，将毛细细毛放在原位。然后将毛细细金银放入光解单元底部的凹槽通道中，并将毛细细金银窗与光解细胞窗口对中心。将毛细细毛放在位，并添加磁性面膜。将照片分析盖放回原位。将毛细管放入圆分细胞底部的凹槽通道中，并将第二个毛细管窗口放在多厘米细胞中心的小孔径上。一手握住毛细细毛，一手将两个磁性夹子放在原位，将毛细细毛放在原位。关闭多米细胞，直到它点击关闭。最后，将毛细金插入产品收集器毛细的扶手上方的刀尖旋钮，将毛细条延伸到小瓶底部的正上方。使用切割机切割所需的特氟隆管长度，并检查末端是否有干净的直切。通过其中一个螺母插入新注射回路的一端，并将新的铁杉放在管子的末端。将螺母和铁杉放在原位，同时将管插入注水阀的端口六，直到其底部。用扳手拧紧螺母，然后确认螺母和铁杉牢固到位。一旦两端有一个螺母和固定的铁杉，松散地拧一端到端口三和另一端端。一旦到位，拧紧两侧手指紧，然后四分之一转过去手指紧用扳手。启动流体模块，打开无激光HRPF系统，然后是光解模块、圆圆模块、产品收集器和系统计算机。启动系统软件。从注射器泵上的阀门位置将管子完全浸入 10 毫升缓冲器中。将管子从废品位置和管子从注射器端口上的二号港引导到一个空容器中收集废物。将 1.5 毫升微中流管放置在产品收集器旋转木马上标有 HN6 的位置。通过注入 25 微升 HPLC 级水，将阀门设置在负载位置，五次冲洗出喷射回路。手动转动喷射阀以冲洗系统其余部分。选择控制软件上的进程，开始流动水，直到水滴形成。制作含有两毫升腺苷和10微摩尔蛋白的溶液。然后使用每毫升卡塔拉塞0.3毫克和35毫摩尔甲基安氨酸做淬淬溶液。将 25 微升淬淬的溶液放入 200 微升微管中。将过氧化氢稀释到200毫升，将其保持在冰上。在控制软件上以零伏启动闪光电压。这种零伏控制将决定兴趣蛋白的任何背景氧化。选择启动数据自动零，然后进行进程然后准备。最后，将喷射阀调低至负载位置。将淬泄解决方案放在产品收集器旋转木马上的位置，然后将产品小瓶更改为系统控制软件上的小瓶。注射前，将12.5微升腺苷和蛋白质混合物与12.5微升过氧化氢混合。将混合物上下混合，然后快速旋转下来。混合后10秒内使用注射端口注射25微升溶液。将注射阀切换到注射位置，并在处理样品时等待。将控制软件上的闪光电压调高至 750 伏特，并重复标记步骤。然后记录每个样本的吸收度。首先，单击"选择"，然后手动选择峰值吸收的开始和结束。在可用空间中，写入示例的描述。对于所有已获得的数据，重复此步骤。将数据复制并粘贴到电子表格中，以计算每个电压腺苷吸收量的平均变化。采集完所有样品后，将注射阀设置为负载位置，将注射阀和样品循环冲出，并注射 25 微升 HPLC 水五次。将注射阀调至喷射位置，用 HPLC 水冲洗系统其余部分。冲洗后，停止流量，退出系统控制软件，并关闭模块，从产品收集器、圆圆模块、光解模块开始，最后是流体模块。氧化后，用于量程的腺苷在265纳米时会降低紫外线吸收量。缓冲区 2 包含一个激进的清理剂，与缓冲区 1 相比，它减少了腺苷吸收的变化。在四个增加的等离子灯电压下，在过氧化氢和腺苷的存在下，对阿波米奥球蛋白进行了改良。检测到六个肽，氧化的线性增加，在265纳米的腺苷吸收变化。这六种氧化肽被标记在肌红蛋白的晶体结构上。从高压等离子灯检测到的氧化程度远远高于激光法。这种增加源于高压等离子灯的广谱紫外线发射光谱。与Kr F Excimer激光和Nd YAG激光相比，高压等离子灯通过产生宽广的紫外光谱，更好地重叠过氧化氢的吸收域，从而通过过氧化氢的光解更有效地生产羟基。高压等离子灯显著提高了羟基基的产量，超过了通常使用 Kr F Excimer 激光观察到的产量。使用100毫摩尔过氧化物可引起高水平的背景氧化。如果蛋白质系统容易受到过氧化物引起的氧化，将浓度降低到10毫升。HRPF 的不可逆标签允许下游样品处理，包括长蛋白酶消化，您可以添加串联质量标签进行多重处理，并执行 2D 色谱，所有这些都可改善肽检测和协调结构信息。

Research

Education

JoVE Journal

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Cell Biology

Biochemistry

Unit 1

Protein Structure

SCIENTISTS IN ACTION

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

结合干湿实验室技术指导大型卷曲螺旋含有蛋白质的结晶

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

蛋白质的表征(按尺寸-排除色谱与多角度光散射相结合(SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Analysis of SEC-SAXS data via EFA deconvolution and Scatter

通过 EFA 去卷和散射分析 SEC-SAXS 数据

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ...

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

使用巴库洛病毒表达载体系统生产重组PRMT蛋白

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. ...

JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics

JUMPn：蛋白质共表达聚类和网络分析在蛋白质组学中的简化应用

With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. ...