Name: a tandem liquid chromatography–mass spectrometry-based approach for metabolite analysis of staphylococcus aureus
Uploaded: 2017-03-28T15:00:00.000+00:00
Duration: 8 min 3 s
Description: Watch this Scientific Journal Video about A Tandem Liquid Chromatographyâ€“Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus at JoVE.com

这项工作是由美国国立卫生研究院途径来独立奖（授予GM 099893）和教师的启动资金，SRB，以及一个研究项目资助（GM授予042219）的部分资助。该资助者在研究设计，数据收集和解释，或提交作品出版的决定没有任何作用。

1.缓冲溶液的制备<ol><li>制备磷酸盐缓冲盐水;通过稀释10倍的PBS中的储备溶液，以1倍的用超纯水的最终浓度（PBS pH 7.4）中（蒸馏水和去离子）水。 </li><li>通过组合2毫升乙腈，2毫升甲醇，1毫升超纯H 2 O和甲酸的19微升（0.1毫摩尔终浓度）制备骤冷溶液。 </li><li>制备LC-MS通过添加甲酸（0.2％[v / v]的终浓度），以超纯水溶剂A。 </li><li>通过添加甲酸（0.2％[v / v]的终浓度），以乙腈制备LC-MS溶剂B。 注:所有的溶液应使用最高纯度的试剂可用（通常高效液相色谱级）来制备。解决方案应准备每次实验前新鲜的和储存到使用前的冰。 </li></ol> 2.稳态金黄色葡萄球菌生长的建立<ol><li>条纹S.黄色葡萄球菌从冷冻甘油股票的胰蛋白酶大豆琼脂（TSA）隔离兴趣小号株。孵育在37℃下16-24小时。 </li><li>接种4毫升的胰蛋白酶大豆肉汤（TSB）或与各菌株的单菌落无菌玻璃试管孵育另一种合适的介质中。孵育倾斜（〜70°角），在37℃下16-20小时在每分钟（rpm）的60转旋转。 注:过夜培养物使用标准方法时，包括在步骤2.2中所述，这会影响细胞生理学容易发生氧梯度。因此，我们采用了多背稀释战略，以确保生物稳态（见步骤2.4-3.2，下同）。 </li><li>使用分光光度计从步骤2.2在600nm（OD 600）测量所述培养物的光密度。使用无菌介质作为光学参考（空白）。稀释这些细胞的0.05在50ml无菌TSB培养基的OD 600（预热至37℃）在单独的，250个毫升烧瓶德朗。 </li><li> INCU软化剂在37℃下与在280rpm下摇动的水浴中培养。 </li><li>每隔30分钟，采取OD 600个测量结果;作为光密度的增加，可能变得需要稀释培养物与TSB，使它们保持分光光度计的线性吸光度范围内。 </li><li>当来自步骤2.5培养物实现的〜0.8-1.0的OD 600时 ，传代培养这些入50mL 37℃的TSB到0.01-0.05的OD 600，并重复步骤2.4和2.5。 </li></ol> 3.样品采集设置<ol><li>制备碎干冰的床在合适的容器（ 例如，玻璃皿，冰桶，或冷却器）。 </li><li>作为培养物的光密度接近期望收获点，添加猝灭溶液到35毫米的未经处理的培养皿中，并在干冰 - 凉爽预为≥5分钟1毫升。 注意:"期望的收获点"将取决于实验的目标有所不同。例如，如果一个人检查米etabolites有氧生长期间，这种状态的关键指标包括醋酸排泄和在指数生长后阶段32，33乙酸盐再同化。通常，这一点应该是一个特定的生长阶段（ 例如，指数期）内。与此级相关联的特定OD 600个值可能不同的细菌菌株和生长介质之间变化。 </li><li>放置一个不锈钢过滤器料（预冷却至-20℃）中的橡胶塞，并把它连接到一个室内真空或真空泵的真空烧瓶的顶部。 </li><li>施加真空并在顶部放置一个混合纤维素酯膜（0.22微米孔径）。 注意:关键是要使用的过滤器的直径等于玻璃料和适当居中此过滤器，以确保在样品通过过滤器，而不是在边缘绘制。用冰冷，无菌水润湿膜2 O可以帮助与定位膜。 </li></ol> 4.样品收获<ol><li>在OD 600〜0.4-0.5，使用一个血清移液管从烧瓶中除去13毫升培养物，并在样品应用到过滤器。 </li><li>整个样品已经被过滤之后，立即清洗过滤用冰冷的PBS的≥5mL至洗去介质相关的代谢物。 </li><li>断开真空，并使用一对无菌镊子从玻璃料除去过滤器。倒置滤波器（电池侧下）插入预冷的淬灭溶液中。 注意:要快速执行上述步骤是非常重要的（ 即，几秒钟内），并一旦液体已被移除，以确保细胞的快速淬火，阻止代谢活性。 </li><li>孵育在干冰上急冷溶液中的滤波器为≥20分钟。 </li><li>使用无菌镊子，反转在陪替氏培养皿的过滤器（细胞的一面朝上），并使用微量吸管冲洗细胞关闭t的他膜进入骤冷溶液。 </li><li>重新悬浮在骤冷液中的细胞，然后将细胞悬浮液转移到含有100〜μL为0.1mm二氧化硅珠的无菌2mL的耐冲击管。存储此在干冰上或在-80℃下。 </li></ol> 5.代谢物提取<ol><li>在湿冰上解冻样品和破坏在均化器将细胞与4 30秒脉冲串以6000rpm，与周期之间干冰2 min的冷却周期。 </li><li>澄清15分钟裂解物中以最大速度预冷，冷藏微量（ 即，在≤4℃下18213×g）离心。 </li><li>将上清转移至干净的微离心管中。 </li><li>使用微量，转移样品到用于在步骤6的残余肽含量定量微量离心管的一小部分;存储在-80℃下的余数。 注:保留样品的体积而变化，这取决于在步骤6.1中使用的BCA测定。这个样品应被存储在立即分析湿冰上或在-80℃冷冻。 </li></ol> 6.二喹啉甲酸（BCA）测定法<ol><li>所推荐的试剂盒制造商，使用来自步骤5.4的样品，以测定每一样品的残余肽浓度进行BCA测定。 </li></ol> 7. LC-MS <ol><li>混合用LC-MS溶剂B的75μL75 S的μL 黄色葡萄球菌提取物，在步骤1.4制备。 </li><li>涡旋混合和旋以13,000×g离心5分钟。 </li><li>放置100μL的上清液的成液相色谱（LC）小瓶中，并且盖住它。确保没有气泡被俘获于样品中。 </li><li> 装入小瓶LC到LC-MS自动进样器和软件中编辑的运行列表"脱机工作列表编辑器"。 <ol><li>填写"样品名称"（ 例如，野生型-1）， "样本位置"（ 例如，P1-A1）， "方法"（ 例如，甲酸甲CID-负的方法），和"数据文件"（ 例如，野生型-1）列。点击按钮"保存工作列表"按钮。打开"质谱数据采集工作站"软件，并输入以前保存的工作列表。点击"开始运行工作列表"按钮，开始连续LC-MS测定。 </li></ol></li><li>分离样品在列，列链接到飞行（TOF）光谱仪的时间，夫妇与LC系统的TOF质谱仪。如下使用移动相梯度:0-2分钟，85％溶剂B; 3-5分钟，80％溶剂B; 6-7分钟，75％溶剂B; 8-9分钟，70％溶剂B; 10-11.1分钟，50％溶剂B; 11.1-14分钟，20％溶剂B;和14.1-24分钟，5％溶剂B;与在85％溶剂B在10分钟再平衡阶段和0.4毫升分钟-1的流速结束。 </li><li> 使用等度泵，注入一个参考质量溶液与运行，以允许同时质量轴校准。 注:此步骤是基于标准TOF光谱仪手册。 <ol><li>使用乙酸D4和六（1H，1H，3H四氟）膦嗪作为参考质量溶液进行实时校准的混合物。使用具有2.5毫升分钟的流速等度泵-1输注。 </li></ol></li></ol> 8.离子计数的批量修正<ol><li>指定的任何样品，以作为批次校正的参考样品（ 例如，野生型，复制1）。 </li><li>计算离子计数的总和的参考样品之内的所有代谢物。重复此计算所有样本。 </li><li>由参考样品的总离子计数将每个样品的总离子计数以产生一个比率。 </li><li>除以样本/参照比值的样品内，用于每种代谢物的离子计数以获得用于每种代谢物的分批校正离子计数。 </li></ol> 9.肽规范化<ol><li> ðivide对于在步骤8中通过与步骤6中的BCA测定法测定，得到的归一化值，每种代谢物的肽浓度而获得的每个样品的批校正离子计数值。 注:在步骤9.1获得的每种代谢物的归一化，批次间歇校正离子计数可菌株之间直接进行比较，并进行统计分析（ 例如，曼- -惠特尼U -test）。可替换地，已知的代谢物以不改变或者通过治疗或遗传背景可以被用作一个归一化器，以检测由于代谢物分解的变化。在提取缓冲液L-正缬氨酸或gluraric酸的已知量的内含物可用于样品处理34期间校正损失。 </li></ol>

作者宣称，他们没有竞争的经济利益。

所有小分子代谢物通过中央的代谢途径的共同起源地相互连接。在指数生长，细菌细胞在生物和代谢稳定状态下，在特定条件下提供所述生理状态的快照。 CODY通过响应ILV和GTP监视养分充分。由于ILV和GTP池下降，科迪活性很可能逐渐减少，调整它的靶基因的表达，以适应增加的养分耗竭30。甲科迪缺陷型菌株的行为就像ILV和GTP从环境耗尽但仅表现出相对生长行为的科迪-精通应变?...

细菌病原体必须与主机环境中的诸多挑战抗衡。除了通过免疫细胞直接攻击时，主机还吸收营养物质对细菌的生存和复制，产生营养免疫力1，2至关重要。为了生存，这些恶劣的环境中，细菌病原体部署致病因子。其中的一些因素使得细菌逃避免疫反应;其他因素包括分泌的消化酶，如透明质酸酶，热核反应的，和脂肪酶，其可以使得细菌通过消耗组织来源的成分3，4，5以补充缺少的营养成分。事实上，细菌已经进化出扎细胞的生理状态到生产毒力因子6,7的调节系统， <s<html>向上类= "外部参照"&gt; 8，9，10。 越来越多的证据指向身体科迪到作为一个关键调节器连接代谢和毒性。虽然在枯草芽孢杆菌首先发现作为二肽通透（DPP）基因11的阻遏，科迪现在已知要产生由几乎所有的低G + C革兰氏阳性菌12,13和调节几十参与碳基因和氮代谢14，15，16，17，18，19。在致病物种，科迪还控制的一些最重要的毒力基因20，21的表达，。EF"&gt; 22，23，24，25，26，27科迪被激活为DNA结合蛋白由两个类配体:支链氨基酸（支链氨基酸;异亮氨酸，亮氨酸，和缬氨酸[ILV]）和GTP当这些营养丰富，科迪压制（或在某些情况下，刺激）转录。由于这些营养物变得有限，科迪活性逐渐降低，导致分级转录应答重新路由通过连接到中心代谢的各种代谢途径的前体28，29，30。 串联耦合到质谱（LC-MS）液相色谱法是一种强大的技术，可以准确地识别和量化小分子的细胞内代谢物31。当与创见配对riptome分析（ 例如，RNA测序），该分析工作流可以提供深入了解发生在响应于环境或营养应激的生理变化。这里，我们提出用于通过LC-MS从金黄色葡萄球菌细胞，并随后分析代谢物的提取的方法。这种方法已被用来证明科迪对金黄色葡萄球菌生理的多效性影响。

<table><tbody><tr><td>&lt;strong&gt;Material/Equipment&lt;sup&gt;a&lt;/sup&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DeLong Culture Flask (250 mL)</td><td>Belco</td><td>2510-00250</td><td></td></tr><tr><td>Sidearm Flask, 500 mL</td><td>Pyrex</td><td>5340</td><td></td></tr><tr><td>3-hole Rubber Stopper, #7</td><td>Fisher</td><td>14-131E</td><td></td></tr><tr><td>Stainless Steel Filter holder/frit</td><td>VWR</td><td>89428-936</td><td></td></tr><tr><td>Petri Dish, 35 mm</td><td>Corning</td><td>430588</td><td>Not tissue culture treated</td></tr><tr><td>Mixed cellulose ester membrane, 0.22 &amp;mu;m pore size</td><td>Millipore</td><td>GSWP02500</td><td></td></tr><tr><td>Impact-resistant tubes, 2 mL</td><td>USA Scientific</td><td>1420-9600</td><td></td></tr><tr><td>Silica Beads, 0.1 mm</td><td>Biospec Products Inc</td><td>11079101Z</td><td></td></tr><tr><td>Precellys 24 homogenizer</td><td>Bertin Instruments</td><td>EQ03119-200-RD000.0</td><td></td></tr><tr><td>Micro BCA Protein Assay Kit</td><td>Pierce (Thermo Scientific)</td><td>23235</td><td></td></tr><tr><td>Cogent Diamond hydride type C column</td><td>Agilent</td><td>70000-15P-2</td><td></td></tr><tr><td>Accurate-Mass Time-of-Flight (TOF) LC-MS, 6200 Series</td><td>Agilent</td><td>G6230B</td><td></td></tr><tr><td>Quat Pump, 1290 Series</td><td>Agilent</td><td>G4204A&amp;nbsp;</td><td></td></tr><tr><td>Bin Pump, 1290 Series</td><td>Agilent</td><td>G4220A&amp;nbsp;</td><td></td></tr><tr><td>Valve Drive, 1290 Series</td><td>Agilent</td><td>G1107A&amp;nbsp;</td><td></td></tr><tr><td>Isocratic Pump, 1290 Series</td><td>Agilent</td><td>G1310B&amp;nbsp;</td><td></td></tr><tr><td>TCC, 1290 Series</td><td>Agilent</td><td>G1316C&amp;nbsp;</td><td></td></tr><tr><td>Sampler, 1290 Series</td><td>Agilent</td><td>G4226A&amp;nbsp;</td><td></td></tr><tr><td>Thermostat, 1290 Series</td><td>Agilent</td><td>G1330B&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Chemical&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tryptic Soy Broth</td><td>Becton Dickinson</td><td>211825</td><td></td></tr><tr><td>Difco Agar, Granulated</td><td>Becton Dickinson</td><td>214530</td><td>Solid media contains 1.5% [w/v] agar</td></tr><tr><td>Phosphate-buffered saline (pH 7.4) 10x</td><td>Ambion</td><td>AM9624</td><td>Dilute fresh to 1x with ultra-pure water</td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific</td><td>A955-500</td><td>Optima LC-MS</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A456-500</td><td>Optima LC-MS; toxic</td></tr><tr><td>Formic Acid</td><td>Sigma Aldrich</td><td>94318</td><td>For mass spectrometry, 98%</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MassHunter</td><td>Agilent</td><td>G3337AA</td><td></td></tr><tr><td>&lt;strong&gt;Bacterial Strain&lt;/strong&gt;</td><td>&lt;strong&gt;Species&lt;/strong&gt;</td><td>&lt;strong&gt;Strain&lt;/strong&gt;</td><td>&lt;strong&gt;Genotype&lt;/strong&gt;</td></tr><tr><td>SRB 337</td><td>Staphylococcus aureus</td><td>USA200 MSSA UAMS-1</td><td>wild type</td></tr><tr><td>SRB 372</td><td>Staphylococcus aureus</td><td>USA200 MSSA UAMS-1</td><td>&amp;Delta;codY::erm</td></tr><tr><td colspan="4">&lt;sup&gt;a&lt;/sup&gt;Chemicals and materials listed are specific to the method described and do not include standard laboratory chemicals or supplies.</td></tr></tbody></table>

a tandem liquid chromatography&#8211;mass spectrometry-based approach for metabolite analysis of staphylococcus aureus

在努力阻止细菌病原体，主机往往限制了营养素在感染部位的可用性。这种限制可以改变，其调控因子做出响应，调节细胞代谢关键代谢物的丰度。近年来，一些蛋白质和RNA已成为致病基因表达的重要调节器。例如，科迪蛋白响应支链氨基酸和GTP的水平并广泛保守的低G + C革兰氏阳性细菌。如金黄色葡萄球菌全球监管机构，科迪控制数十种毒性和代谢基因的表达。我们推测， 金黄色葡萄球菌使用CODY，部分地改变在努力适应宿主环境可能遇到的营养限制条件的代谢状态。这个手稿描述了使用加上质谱液相色谱法从金黄色葡萄球菌提取并分析代谢物的方法trometry，开发是为了检验这一假设的协议。该方法还强调，将确保严密性和可重复性的最佳做法，如维持生物稳态和不断充气，无需使用连续恒化培养。相对于USA200甲氧西林敏感的金黄色葡萄球菌分离UAMS-1亲本菌株，同基因的突变体科迪表现出从天门冬氨酸（ 例如 ，苏氨酸和异亮氨酸）衍生的氨基酸显著增加和它们的前体下降（ 例如 ，天冬氨酸和O- -acetylhomoserine ）。这些发现与RNA-SEQ分析获得的转录数据良好相关:基因在这些途径均在科迪无效突变体10和800倍之间上调。耦合转录组和代谢组的整体分析可以揭示细菌如何改变它们的代谢，当面对的环境或营养应激，提供潜在洞察的PHY与养分枯竭相关iological变化感染期间经历。这些发现可能铺平新型抗感染药物和治疗学的发展道路。

在一个丰富的，复杂的媒体在体外生长过程中我们分析了金黄色葡萄球菌胞内代谢库。作为原则证明，我们比较了甲氧西林敏感的金黄色葡萄球菌骨髓炎之间代谢特征隔离UAMS-1（野生型WT]）和缺少全局转录调节科迪（Δ 科迪 ）26的等基因菌株。稳态中，WT和Cody菌株的指数培养物建立于TSB培养基，如在该协议的步骤2中描述...

Watch this Scientific Journal Video about A Tandem Liquid Chromatographyâ€“Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus at JoVE.com

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

在这里，我们描述了用于通过液相色谱法和质谱分析法从金黄色葡萄球菌和它们的后续分析的代谢物的提取的协议。

对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em&gt;金黄色葡萄球菌</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

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Biochemistry

10.0K 次观看.  Georgetown University. 在这里，我们描述了用于通过液相色谱法和质谱分析法从金黄色葡萄球菌和它们的后续分析的代谢物的提取的协议。 

视频: A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

规模以上非靶向代谢组学剖析血清超高效液相色谱 - 质谱（UPLC-MS）

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

科研

化学

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

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 ...

生物化学

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of NPAAs can provide crucial information about cellular uptake and/or function, metabolic pathways, and potential toxicity. &#946;-methylamino-L-alanine (BMAA) is a neurotoxic NPAA produced by various algae species and is associated with an increased risk for neurodegenerative diseases, which has led to significant research interest. There are numerous ways to extract AAs for analysis, with liquid chromatography-tandem mass spectrometry being the most common, requiring protein precipitation followed by acid hydrolysis of the protein pellet. Studies on the presence of BMAA in algal species provide contradictory results, with the use of unvalidated sample preparation/extraction and analysis a primary cause. Like most NPAAs, protein precipitation in 10% aqueous TCA and hydrolysis with fuming HCl is the most appropriate form of extraction for BMAA and its isomers aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB). The present protocol describes the steps in a validated NPAA extraction method commonly used in research and teaching laboratories.

The present protocol describes the extraction of non-protein amino acids from biological matrices via trichloroacetic acid (TCA) protein precipitation and acid hydrolysis before analysis using liquid chromatography-tandem mass spectrometry.

从蓝藻中提取非蛋白质氨基酸用于液相色谱-串联质谱分析

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of ...

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

基于莱萨特的无细胞系统中的代谢物分析液色谱耦合折射指数或质谱检测

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...

生物工程

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

敏感，大规模定量代谢组学的策略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

生物学

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an ...

对于代谢物分析基于法 - 串联液相色谱 - 质谱法

摘要

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此视频中的章节

规模以上非靶向代谢组学剖析血清超高效液相色谱 - 质谱（UPLC-MS）

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

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

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

使用液体色谱加上串联质谱法的糖核酸菌的定量元细胞学

从蓝藻中提取非蛋白质氨基酸用于液相色谱-串联质谱分析

基于莱萨特的无细胞系统中的代谢物分析液色谱耦合折射指数或质谱检测

敏感，大规模定量代谢组学的策略

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

对于代谢物分析基于法 - 串联液相色谱 - 质谱法

摘要

探索更多视频

此视频中的章节

规模以上非靶向代谢组学剖析血清超高效液相色谱 - 质谱（UPLC-MS）

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

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

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

从蓝藻中提取非蛋白质氨基酸用于液相色谱-串联质谱分析

基于莱萨特的无细胞系统中的代谢物分析液色谱耦合折射指数或质谱检测

敏感，大规模定量代谢组学的策略

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

使用液体色谱加上串联质谱法的糖核酸菌的定量元细胞学