Name: a tandem liquid chromatography&#8211;mass spectrometry-based approach for metabolite analysis of staphylococcus aureus
Uploaded: 2017-03-28T15:00:00
Description: Watch this Scientific Journal Video about A Tandem Liquid ChromatographyÃ¢â‚¬â€œMass 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...

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 border="0" cellpadding="0" cellspacing="0" width="933">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Material/Equipmenta</td>
			<td style="width:167px;"></td>
			<td style="width:163px;"></td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">DeLong Culture Flask (250 ml)</td>
			<td style="width:167px;">Belco</td>
			<td>2510-00250</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Sidearm Flask, 500 ml</td>
			<td style="width:167px;">Pyrex</td>
			<td style="width:163px;">5340</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">3-hole Rubber Stopper, #7</td>
			<td style="width:167px;">Fisher</td>
			<td style="width:163px;">14-131E</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Stainless Steel Filter holder/frit</td>
			<td style="width:167px;">VWR</td>
			<td>89428-936</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Petri Dish, 35 mm</td>
			<td style="width:167px;">Corning</td>
			<td style="width:163px;">430588</td>
			<td>Not tissue culture treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Mixed cellulose ester membrane, 0.22 &#956;m pore size</td>
			<td style="width:167px;">Millipore</td>
			<td>GSWP02500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Impact-resistant tubes, 2 ml</td>
			<td style="width:167px;">USA Scientific</td>
			<td style="width:163px;">1420-9600</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Silica Beads, 0.1 mm</td>
			<td style="width:167px;">Biospec Products Inc</td>
			<td>11079101Z</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:363px;">Precellys 24 homogenizer</td>
			<td style="width:167px;">Bertin Instruments</td>
			<td style="width:163px;">EQ03119-200-RD000.0</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Micro BCA Protein Assay Kit</td>
			<td style="width:167px;">Pierce (Thermo Scientific)</td>
			<td style="width:163px;">23235</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Cogent Diamond hydride type C column</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">70000-15P-2</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Accurate-Mass Time-of-Flight (TOF) LC-MS, 6200 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G6230B</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Quat Pump, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G4204A&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Bin Pump, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G4220A&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Valve Drive, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G1107A&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Isocratic Pump, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G1310B&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">TCC, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G1316C&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Sampler, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G4226A&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Thermostat, 1290 Series</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G1330B&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td style="width:167px;">Company</td>
			<td style="width:163px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Chemical</td>
			<td style="width:167px;"></td>
			<td style="width:163px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Tryptic Soy Broth</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:163px;">211825</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Difco Agar, Granulated</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:163px;">214530</td>
			<td>Solid media contains 1.5% [w/v] agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Phosphate-buffered saline (pH 7.4) 10X</td>
			<td style="width:167px;">Ambion</td>
			<td style="width:163px;">AM9624</td>
			<td>Dilute fresh to 1X with ultra-pure water</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Acetonitrile</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:163px;">A955-500</td>
			<td>Optima LC-MS</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Methanol</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:163px;">A456-500</td>
			<td>Optima LC-MS; toxic</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">Formic Acid</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:163px;">94318</td>
			<td>For mass spectrometry, 98%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td style="width:167px;">Company</td>
			<td style="width:163px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Software</td>
			<td style="width:167px;"></td>
			<td style="width:163px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:363px;">MassHunter</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:163px;">G3337AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Bacterial Strain</td>
			<td style="width:167px;">Species</td>
			<td style="width:163px;">Strain</td>
			<td>Genotype</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:363px;">SRB 337</td>
			<td style="width:167px;">Staphylococcus aureus</td>
			<td style="width:163px;">USA200 MSSA UAMS-1</td>
			<td>wild type</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:363px;">SRB 372</td>
			<td style="width:167px;">Staphylococcus aureus</td>
			<td style="width:163px;">USA200 MSSA UAMS-1</td>
			<td>&#916;codY::erm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:933px;">aChemicals 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 ChromatographyÃ¢â‚¬â€œMass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus at JoVE.com

A Tandem Liquid ChromatographyÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass 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 ...

Research

JoVE Journal

Biochemistry

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

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

科研

生物化学

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

利用多功能生化方法鉴定新型 ck2 激酶基材

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

一种可定制的酶化生产与纯化天然产物的方法

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

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

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

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 ...

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

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 ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...