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）级试剂尽可能用ACS级或更高的一切存在分析过程中减少干扰的污染物。 1.微流体装置的制备<ol><li>聚甲基丙烯酸甲酯微流控平台的构建<ol><li>获得标准PMMA嵌段（8.9 X 3.8×0.6厘米）和激光烧蚀的输入信道用于引入试剂，一个PROTEO裂解室，并且使用激光雕刻机17，18的输出信道。 注:蚀刻该蛋白水解室中的细长的椭圆形（30×5×0.05mm）的。输入和输出通道必须延伸到PMMA嵌段的端部和不大于75微米的宽度和深度，以便容纳一个30 GA毛细管。 </li><li>切30 GA不锈钢金属毛细管导入的大约10cm每两片使用旋转工具带有一个1/64&quot; 厚切断光盘，使用砂纸来平滑毛细管（这可以通过下的观察来促进的端部光显微镜）。 </li><li>熔体使用烙铁毛细血管进入蚀刻的聚（甲基丙烯酸甲酯）嵌段。输入通道将被连接到自动注射泵，并且输出信道将被用于耦合到MS。 </li></ol></li><li>连续流动的时间分辨动力学调音台的建设<ol><li>获得融合硅玻璃毛细管（ID:75微米，外径:150微米）的约40厘米，并把它插入到大约15厘米的28 GA不锈钢金属毛细管。根据所需的反应时间，使用较长的外金属毛细管或一个具有较大ID。 注:确定金属毛细管的"真"的内径是用于获得精确的反应时间是至关重要的。这可以通过将金属毛细管附接到HPLC来完成，流溶剂虽然毛细管，并记录该背压。甲背压内径计算可以被做，和所确定的金属毛细管的真实内径。这可以通过使用分子量计算器软件（for Windows版本6.49）来计算。 </li><li>产生在内部玻璃毛细管的一端使用低功率激光雕刻机设置的2mm切口和密封内玻璃毛细管（ 图2）的这一端。可替代地，使用陶瓷玻璃毛细使缺口ÿ刀具或用细切割刀片旋转磨床。 </li><li>阵容的内玻璃毛细管与金属毛细管的末端（这可以通过在光学显微镜下观察来促进）。 </li><li>附加此动能混合器混合三通的一端（ 图2）。 </li><li>附加一个熔凝石英玻璃毛细管（ID:75微米，外径:150微米）的大约40厘米到上混合三通动力学混合器的相对端。这被用于递送酸（5％乙酸，pH为2.4），以淬灭反应。 注意:反应物是使用自动输注泵通过聚四氟乙烯（PTFE）管供给到与气密注射器设备。 </li></ol></li><li>胃蛋白酶激活<ol><li>从猪胃粘膜中称出20毫克的胃蛋白酶和在1mL偶联缓冲液（0.1M磷酸钠，0.15M NaCl，pH为6.0）的挂起。 </li><li>称出50毫克N-羟基琥珀酰亚胺（NHS）活化的琼脂糖珠，加入到再-Suspended蛋白酶和轻轻旋转过夜，在4℃。 </li><li>降速在1,000×g离心2分钟，在室温下以收集树脂。 </li><li>吸出未结合的蛋白酶。 </li><li>孵育的琼脂糖在1mL阻断缓冲液（1M的Tris-HCl，pH值6.0）中，并在室温下轻轻转动1个小时。 </li><li>降速在1,000×g离心2分钟，在室温下以收集树脂。 </li><li>吸出封闭缓冲液。 </li><li>孵育胃蛋白酶 - 琼脂糖用1 5毫升％乙酸，5分钟的pH为2.4。 </li><li>降速在1,000×g离心2分钟以收集树脂。 </li><li>吸出上清液。 </li><li>重复步骤1.3.8 - 1.3.10总共三次清洗。 </li><li>存储在1个毫升乙酸珠在4℃下长期使用。 </li></ol></li><li>设备装配<ol><li>填充的使用无菌刮刀在5％乙酸活化胃蛋白酶 - 琼脂糖珠浆液中的蛋白质水解室中。 </li><li>放置PMMA微platfor米的两个空白PMMA嵌段作为覆盖密封装置之间，衬有硅橡胶以产生液密密封。 </li><li>使用金属夹板到压力密封装置。 注:在夹具以适合微流体平台被定制，并且由两个板测量10.1厘米X7.4厘米×1.2厘米。 </li><li>以10μL/ min的速率流过5％乙酸，pH为2.4。以1微升/分钟的速率流50 mM乙酸铵缓冲液（pH 6.9），尽管该蛋白质线。 注意:这是极为重要的是，乙酸被连续地流过该装置在整个实验的全部。保证不存在任何泄漏和使得流体从所述输出通道，其将用作ESI源仅离开。 </li><li>耦合装置的改性四极时间飞行（Q-TOF）质谱使用可调绝缘阶段，以达到最佳电条件光谱仪的前端。 注:旁路开关为了模拟商业ESI源的存在下被引入。 17 </li></ol></li></ol> 2.时间分辨电喷雾电离氢氘交换<ol><li>胃蛋白酶的采集和蛋白质只有光谱 注:ESI-MS采集以正离子模式与4500的电压进行，以5000，60-V去簇电势，和250-V聚焦电势。谱获取在一定范围350-1,500的m / z为1个s的扫描速率-1。 <ol><li>获得胃蛋白酶只是谱。一旦蛋白质添加任何出现在该光谱峰减去。 </li><li>以1微升/分钟，其中50 mM乙酸铵缓冲液先前被流动的速率引入50-100μMTAU /磷酸-τ蛋白（净化和如先前所描述13制备，19）。 </li><li>获得的蛋白质谱只。 </li></ol></li><li>收购Ø˚F时间点<ol><li>而100μM的TAU /磷酸-τ蛋白在1μL/ min的速度流动，在通过T形连接件的3微升/分钟的速率引入D 2 O中，并允许在动力学混合器进行反应。允许系统采集的频谱之前平衡至少10分钟。 发生在蛋白水解室中的交换之后，在标记反应通过乙酸pH为2.4的10微升/分钟的标记的蛋白质的流动和消化猝灭:注意。 </li><li>为了提高标记时间，手动拉回内玻璃毛细管的位置，以获得混合的42毫秒倍至8秒。允许系统中的每个回拉之间，以平衡至少10分钟。 </li></ol></li></ol> 3.数据和统计分析<ol><li>识别肽和计算氘交换的百分比<ol><li>进行质谱分析使用mMass软件，版本5.5.0 20 </suP&gt;。 </li><li>识别使用自ExPASy蛋白质组学FindPept服务器肽和所需的21当通过碰撞诱导解离（CID）确认。 注:在这里，氘吸收合并使用一个内部开发FORTRAN软件进行同位素分布分析22，23测量。 </li><li>计算基于使用SPHERE网络工具22，24的一级序列的理论内在利率。 </li><li>使用单指数非线性回归拟合数据，并使用图形和统计软件（ 例如 ，的SigmaPlot）正常化。 注意:所述的比率k INT / K OBS产生的保护因子（PF），该特定区域是在构象合奏内结构化程度的半定量测量。 </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+and+Type+2+scenarios+in+hydrogen+exchange+mass+spectrometry+studies+on+protein-ligand+complexes.">Type 1 and Type 2 scenarios in hydrogen exchange mass spectrometry studies on protein-ligand complexes.</a> The Analyst. 139, 6078-6087 (2014).</li></ol>

We have nothing to disclose.

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><tbody><tr><td>Poly(methyl methacrylate) or PMMA</td><td>Professional Plastics</td><td>SACR.250CCP</td><td>8.9 cm x 3.8 cm x 0.6 cm</td></tr><tr><td>Fused Silica Glass Capillary</td><td>Polymicro Technologies</td><td>106815-0018</td><td>ID: 75 &amp;micro;m, OD: 150 &amp;micro;m</td></tr><tr><td>Metal Capillaries</td><td>McMaster-Carr</td><td>28 ga &amp;ndash; 89875K97&lt;br/&gt; 30 ga&amp;nbsp; - 89875K99</td><td></td></tr><tr><td>Fluorinated Ethylene Propylene (FEP) Tubing</td><td>IDEX</td><td>1477&lt;br/&gt; 1548</td><td>ID: 0.007&amp;rdquo;, OD: 1/16&amp;rdquo;&lt;br/&gt; ID: 0.020&amp;rdquo;, OD: 1/16&amp;rdquo;</td></tr><tr><td>Standard Polymer Tubing Cutter</td><td>IDEX</td><td>A-327</td><td>for 1/16&amp;rdquo; and 1/8&amp;rdquo; OD tubing</td></tr><tr><td>Micro Static Mixing Tee</td><td>IDEX</td><td>M-540</td><td>for 1/16&amp;rdquo; OD tubing</td></tr><tr><td>or</td><td></td><td></td><td></td></tr><tr><td>Stainless Steel Tee, 0.25&amp;nbsp;mm Bore</td><td>Valco Instruments Co., Inc. (VICI)</td><td>ZT1C</td><td>for 1/16&amp;rdquo; OD tubing</td></tr><tr><td>PEEK Tee for 1/16&amp;rdquo; OD Tubing</td><td>IDEX</td><td>P-727</td><td></td></tr><tr><td>10-32 Female to Female Luer</td><td>IDEX</td><td>P-659</td><td></td></tr><tr><td>10-32 PEEK Double-Winged Nut</td><td>IDEX</td><td>F-300</td><td></td></tr><tr><td>Ferrule for 1/16&amp;rdquo; OD Tubing</td><td>IDEX</td><td>F-142</td><td></td></tr><tr><td>100 Series Rotary Tool</td><td>Dremel</td><td>F013010001</td><td></td></tr><tr><td>Cut-Off Discs</td><td>Jobmate</td><td></td><td>1/64&amp;rdquo; thickness</td></tr><tr><td>Stereomaster Digital Zoom Microscope</td><td>Fisher Scientific</td><td>12-563-411</td><td></td></tr><tr><td>Soldering Iron</td><td>Mastercraft</td><td>58-6301-2</td><td></td></tr><tr><td>VersaLaser</td><td>Universal Laser</td><td></td><td></td></tr><tr><td>Syringes</td><td>Hamilton</td><td>81220</td><td>500&amp;nbsp;&amp;micro;L capacity</td></tr><tr><td>Syringe Pumps</td><td>Harvard Apparatus</td><td>70-4501</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NHS-Activated Agarose</td><td>Fisher Scientific</td><td>26196</td><td></td></tr><tr><td>Pepsin from Porcine Gastric Mucosa</td><td>Sigma-Aldrich</td><td>P6887-250MG</td><td></td></tr><tr><td>Deuterium Oxide</td><td>Sigma-Aldrich</td><td>151882-10X0.6ML</td><td></td></tr><tr><td>Acetic Acid</td><td>Sigma-Aldrich</td><td>695092-100ML</td><td></td></tr><tr><td>HPLC Grade Water</td><td>Fisher Scientific</td><td>W5-4</td><td></td></tr><tr><td>Ammonium Acetate</td><td>Sigma-Aldrich</td><td>A7330-500G</td><td></td></tr><tr><td>Sodium Phosphate</td><td>Fisher Scientific</td><td>S369-500</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>S671-3</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software/Online Tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CorelDraw X3</td><td>Corel</td><td></td><td></td></tr><tr><td>Molecular Weight Calculator</td><td></td><td>Version 6.49</td><td>Open Source MS Tool</td></tr><tr><td>mMass</td><td></td><td>Version 5.5.0</td><td>Open Source MS Tool</td></tr><tr><td>ExPASy FindPept</td><td>Swiss Institute of Bioinformatics</td><td></td><td></td></tr><tr><td>SigmaPlot</td><td>Systat Software</td><td>Version 11.0</td><td></td></tr><tr><td>PyMOL</td><td>Schr&amp;ouml;dinger</td><td>Version 1.5.0.4</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>QStar Elite Hybrid Q-TOF Mass Spectrometer</td><td>AB SCIEX</td><td></td><td></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. ...

time-resolved-electrospray-ionization-hydrogen-deuterium-exchange

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

9.8K 次观看.  York University. 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。 

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

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

使用氢交换质谱分析蛋白质动力学

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

科研

化学

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

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

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 ...

生物化学

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

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 virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

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 ...

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

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality for: (1) The identification of related protein structure sets to user specified thresholds of similarity; (2) Their multiple alignment and structure superposition; (3) Sequence and structure conservation analysis; (4) Inter-conformer relationship mapping with principal component analysis, and (5) comparison of predicted internal dynamics via ensemble normal mode analysis. This integrated functionality provides a complete online workflow for investigating sequence-structure-dynamic relationships within protein families and superfamilies.

A protocol for the online investigation of protein sequence-structure-dynamics relationships using Bio3D-web is presented.

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 ...

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

质谱途径研究蛋白质结构和相互作用的冻干粉

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

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

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

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 ...

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

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics&#8722;even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

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

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

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 ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

基于毛细管电泳的氢/氘交换，用于自上而下的质谱法对蛋白质进行构象表征

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

高分辨率中子光谱研究蛋白质和水合水的皮秒纳秒动力学

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

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

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...