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。

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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

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

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 ...

科研

生物化学

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 ...

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 ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

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 ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

叶喷雾质谱: 一种快速的环境电离技术直接评价植物组织中的代谢产物

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

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 ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

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 ...

Quaternary Structure Modeling Through Chemical Cross-Linking Mass Spectrometry: Extending TX-MS Jupyter Reports

Protein-protein interactions can be challenging to study yet provide insights into how biological systems function. Targeted cross-linking mass spectrometry (TX-MS), a method combining quaternary protein structure modeling and chemical cross-linking mass spectrometry, creates high-accuracy structure models using data obtained from complex, unfractionated samples. This removes one of the major obstacles to protein complex structure analysis because the proteins of interest no longer need to be purified in large quantities. Cheetah-MS web server was developed to make the simplified version of the protocol more accessible to the community. Considering the tandem MS/MS data, Cheetah-MS generates a Jupyter Notebook, a graphical report summarizing the most important analysis results. Extending the Jupyter Notebook can yield more in-depth insights and better understand the model and the mass spectrometry data supporting it. The technical protocol presented here demonstrates some of the most common extensions and explains what information can be obtained. It contains blocks to help analyze tandem MS/MS acquisition data and the overall impact of the detected XLs on the reported quaternary models. The result of such analyses can be applied to structural models that are embedded in the notebook using NGLView.

Targeted cross-linking mass spectrometry creates quaternary protein structure models using mass spectrometry data acquired using up to three different acquisition protocols. When executed as a simplified workflow on the Cheetah-MS web server, the results are reported in a Jupyter Notebook. Here, we demonstrate the technical aspects of how the Jupyter Notebook can be extended for a more in-depth analysis.

通过化学交联质谱法进行四元结构建模:扩展 TX-MS Jupyter 报告

Protein-protein interactions can be challenging to study yet provide insights into how biological systems function. Targeted cross-linking mass ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

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 ...