Name: capturing actively produced microbial volatile organic compounds from human-associated samples with vacuum-assisted sorbent extraction
Uploaded: 2022-06-01T12:30:00
Description: Watch this Scientific Journal Video about Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction at JoVE.com

我们感谢希瑟·毛恩（Heather Maughan）和琳达·M·卡利金（Linda M. Kalikin）对这份手稿的精心编辑。这项工作得到了NIH NHLBI的支持（拨款5R01HL136647-04）。

1. 顶空吸附笔（HSP）和样品分析注意事项
注意：选择含有吸附剂Tenax TA的HSP是为了捕获各种挥发物。与其他吸附剂相比，Tenax对水的亲和力较低，这使其能够从高湿度样品中捕获更多的VOC。Tenax还具有低水平的杂质，可以调节以重复使用。在GC-MS中安装色谱柱时，还考虑了吸附剂的选择（参见 材料表）。
<ol><li>通过提取具有与样品提取相同的条件提取培养基和/或...

<ol>
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L., Pandya, H., Hubbard, M., Turner, M. A., Monks, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GC-MS+analysis+of+ethanol+and+other+volatile+compounds+in+micro-volume+blood+samples-quantifying+neonatal+exposure.">GC-MS analysis of ethanol and other volatile compounds in micro-volume blood samples-quantifying neonatal exposure.</a> Analytical and Bioanalytical Chemistry. 405 (12), 4139-4147 (2013).</li><li>Mayor, A. S. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Headspace+SPME+method+development+for+the+analysis+of+volatile+polar+residual+solvents+by+GC-MS.">Headspace SPME method development for the analysis of volatile polar residual solvents by GC-MS.</a> Journal of Pharmaceutical and Biomedical Analysis. 23 (1), 197-210 (2000).</li><li>Charry-Parra, G., DeJesus-Echevarria, M., Perez, F. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacuum-assisted+sorbent+extraction:+An+analytical+methodology+for+the+determination+of+ultraviolet+filters+in+environmental+samples.">Vacuum-assisted sorbent extraction: An analytical methodology for the determination of ultraviolet filters in environmental samples.</a> Talanta. 208, 120390 (2020).</li><li>Mollamohammada, S., Hassan, A. 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为了鉴定 体外 培养物和人相关样品中的挥发性产生，对 铜绿假单胞菌，金黄色葡萄球菌 和 鲍曼氏 菌的单培养物和共培养物进行挥发性分析，并对不同生物样品进行稳定同位素探测。在单培养和共培养物的分析中，通过在70°C下进行短萃取1小时来检测挥发物。 对单一培养和共培养物的挥发性分析允许对单个物种及其与其他物种相互作用期间产生的化合物进行调查。不同文化...

挥发性有机化合物（VOCs）在细菌检测和鉴定方面具有很大的前景，因为它们是从所有生物体中排放的，并且不同的微生物具有独特的VOC特征。挥发性分子已被用作检测各种呼吸道感染的非侵入性测量，包括慢性阻塞性肺病1，尿中的结核病2和呼吸机相关肺炎4，此外还区分囊性纤维化（CF）受试者和健康对照受试者5，6。挥发性特征甚至已被用于区分CF中的特定病原体感染（金黄色葡萄球菌7，铜绿假单胞菌8，9和金黄色葡萄球菌与铜绿假单胞菌10）。然而，由于这种生物样品的复杂性，通常很难确定特定挥发性有机化合物的来源。
从多种感染微生物中分离挥发性特征的一种策略是对单培养和共培养11中的微生物进行顶空分析。顶空分析检查排放到样品上方“顶空”中的分析物，而不是嵌入样品本身的分析物。微生物代谢物通常在单一培养物中表征，因为在复杂的临床样品中难以确定微生物代谢物的来源。通过分析来自细菌单培养物的挥发物，微生物 在体外 产生的挥发物类型可能代表其挥发性库的基线。结合细菌培养物， 例如，创建共培养物，并分析产生的挥发性分子可以揭示细菌12之间的相互作用或交叉喂养。
鉴定挥发性分子微生物来源的另一种策略是提供用稳定同位素标记的营养来源。稳定同位素是天然存在的，具有不同数量中子的原子的非放射性形式。自20世纪30年代初以来，一种用于追踪动物活性代谢的策略13中，微生物以标记的营养来源为食，并将稳定同位素纳入其代谢途径。最近，重水（D2O）形式的稳定同位素已被用于鉴定临床CF痰液样本14中代谢活性的金黄色葡萄球菌。在另一个例子中，13个C标记的葡萄糖已被用于证明铜绿假单胞菌和Rothia mucilaginosa12的CF临床分离物之间代谢物的交叉喂养。
随着质谱技术的进步，检测挥发性线索的方法已经从定性观察转向更定量的测量。通过使用气相色谱质谱（GC-MS），大多数实验室或临床环境都能够处理生物样品。许多调查挥发性分子的方法已被用于分析样品，例如食品，细菌培养物和其他生物样品，以及空气和水以检测污染。然而，具有高通量的几种常见挥发性取样方法需要溶剂，并且不能以真空萃取提供的优点进行。此外，分析通常需要更大体积或数量（大于0.5 mL）的样品材料15，16，17，18，19，尽管这是底物特异性的，需要针对每种样品类型和方法进行优化。
在这里，采用真空辅助吸附剂提取（VASE）然后在GC-MS上进行热解吸，以调查细菌单培养物和共培养物的挥发性分布，并鉴定来自人类粪便，唾液，污水和痰液样品的稳定同位素探测的活性挥发物（图1）。在样品量有限的情况下，从低至15μL的痰中提取VOCs。对人类样品的同位素探测实验需要添加稳定的同位素源，如 13°C葡萄糖和培养基来培养微生物群落的生长。挥发物的活性生产被GC-MS确定为更重的分子。在静态真空下提取挥发性分子能够以更高的灵敏度检测挥发性分子20，21，22。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>13C glucose</td>
			<td>Sigma-Aldrich</td>
			<td>389374-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Stg Diaph Pump</td>
			<td>Entech Instruments</td>
			<td>01-10-20030</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL VOA vials</td>
			<td>Fisher Scientific</td>
			<td>5719110</td>
			<td></td>
		</tr>
		<tr>
			<td>24 mm Black Caps with hole, no septum</td>
			<td>Entech Instruments</td>
			<td>01-39-76044B</td>
			<td>holds lid liner in place on vial</td>
		</tr>
		<tr>
			<td>24 mm vial liner for sorbent pens</td>
			<td>Entech Instruments</td>
			<td>SP-L024S</td>
			<td>allows pens to make a vacuum seal at top of vial</td>
		</tr>
		<tr>
			<td>5600 Sorbent pen extraction unit (SPEU)</td>
			<td>Entech Instruments</td>
			<td>5600-SPES</td>
			<td>5600 Sorbent Pen Extraction Unit -120 VAC</td>
		</tr>
		<tr>
			<td>96-well assay plates</td>
			<td>Genesee</td>
			<td>25-224</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion (BHI) media</td>
			<td>Sigma-Aldrich</td>
			<td>53286-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemStation Stofware</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DB-624 column</td>
			<td>Agilent</td>
			<td>122-1364E</td>
			<td>60 m, 0.25 mm ID, 1.40 micron film thickness, in GC-MS</td>
		</tr>
		<tr>
			<td>Deuterium oxide</td>
			<td>Sigma-Aldrich</td>
			<td>151882-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexsi sofware</td>
			<td>Dexsi (open source)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GC-MS (7890A GC and 5975C inert XL MSD with Triple-Axis Detector)</td>
			<td>Agilent</td>
			<td></td>
			<td>7890A GC and 5975C inert XL MSD with triple-axis detector</td>
		</tr>
		<tr>
			<td>Headspace Bundle HS-B01, 120VA</td>
			<td>Entech Instruments</td>
			<td>SP-HS-B01</td>
			<td>Items for running headspace extraction included in bundle</td>
		</tr>
		<tr>
			<td>Headspace sorbent pen (HSP) - blank</td>
			<td>Entech Instruments</td>
			<td>SP-HS-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Headspace sorbent pen (HSP) Tenax TA (35/60 Mesh)</td>
			<td>Entech Instruments</td>
			<td>SP-HS-T3560</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (2 mL)</td>
			<td>VWR</td>
			<td>53550-792</td>
			<td></td>
		</tr>
		<tr>
			<td>O-rings</td>
			<td>Entech Instruments</td>
			<td>SP-OR-L024</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Preparation Rail</td>
			<td>Entech Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorbent pen thermal conditioner</td>
			<td>Entech Instruments</td>
			<td>3801-SPTC</td>
			<td></td>
		</tr>
		<tr>
			<td>Todd Hewitt (TH) media</td>
			<td>Sigma</td>
			<td>T1438-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

capturing actively produced microbial volatile organic compounds from human-associated samples with vacuum-assisted sorbent extraction

来自生物样品的挥发性有机化合物（VOC）来源不明。挥发性有机化合物可能来自宿主或宿主微生物群落内的不同生物体。为了解开微生物VOC的起源，对 金黄色葡萄球菌、铜绿假单胞菌 和 鲍曼不动杆菌的细菌单培养物和共培养物进行了挥发性顶空分析，并在粪便、唾液、污水和痰液的生物样品中进行了稳定同位素探测。单培养和共培养用于鉴定单个细菌物种的挥发性产生，或与稳定同位素探测结合使用，以鉴定生物样品中微生物的活性代谢。
采用真空辅助吸附剂萃取（VASE）提取VOCs。VASE是一种易于使用，商业化，无溶剂的顶空提取方法，用于半挥发性和挥发性化合物。与其他提取选项（如 叔丁基化和固相微萃取）相比，提取过程中使用的溶剂和近真空条件使得开发方法相对容易和快速。此处描述的工作流程用于识别来自单一培养和共培养物的特定易失性特征。此外，对人类相关生物样品的稳定同位素探测的分析确定了通常或独特生产的VOC。本文结合活性微生物培养物的稳定同位素探测，提出了VASTE的一般工作流程和实验注意事项。

金黄色葡萄球菌、铜绿假单胞菌和鲍曼氏菌的单一和共培养 单一培养和共培养物由细菌种类 金黄色葡萄球菌、铜绿假单胞菌 和 鲍曼氏菌组成。这些是在人类伤口和慢性感染中发现的常见机会性病原体。为了鉴定单培养物和共培养物中存在的挥发性分子，在70°C下以200rpm搅拌进行短的...

Watch this Scientific Journal Video about Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction at JoVE.com

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

该协议描述了使用真空辅助吸附剂提取方法从生物样品中提取挥发性有机化合物，使用Entech样品制备导轨进行气相色谱结合质谱分析以及数据分析。它还描述了生物样品的培养和稳定同位素探测。

通过真空辅助吸附剂提取从人类相关样品中捕获活性产生的微生物挥发性有机化合物

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

该协议使我们能够轻松地浓缩和鉴定各种生物样品中微生物的挥发性代谢物和活性产生的挥发性挥发物。VASE是一种更人性化的浓缩低丰度挥发物的方法。您所需要的只是在近真空下提供一个样本，剩下的就让物理学来做。获得临床样品的挥发性分析对于在许多不同的疾病状态下发现代谢生物标志物具有重要意义。例如，病原体驱动和感染或抗菌治疗的成功可以通过唾液，痰液或呼吸样本的挥发性分析来检测。为了制备粪便样品，在1.5毫升微量离心管中向100毫克粪便中加入一毫升去离子水并涡旋三分钟。不使用时将样品保存在冰上。将485毫升含有20毫摩尔13C葡萄糖的BHI培养基或含有30%氘的BHI加入15微升粪便和水混合物中，确保样品的最终体积为500微升。准备技术一式三份的样品。为了制备污水样品，将500微升污水加入500微升含有13C葡萄糖或30%氘的BHI培养基中，总体积为1毫升。准备一式三份的样品，并将它们保存在冰上。为了制备唾液样品，将50微升唾液加入500微升BHI培养基中，其中含有13C葡萄糖或30%氘，总体积为550微升。准备一式三份的样品，并将它们保存在冰上。为了制备痰液样品，将15微升痰液加入小瓶中。准备一式三份的样品，并将它们保存在冰上。将空的VOA小瓶放在冷板上，并将冷板放在生物安全罩中的冰上。打开 5600 SPEU 并将其调整到所需温度。使用防水标记，根据样品，重复和HSP ID标记20毫升VOA小瓶，该标记物在冰上时在小瓶外部形成冷凝时可防水。在生物安全罩内，拧下小瓶上的白色盖子，快速将样品移液到小瓶中，然后组装盖子衬垫，黑色盖子和HSP。将含有样品和HSP的小瓶放回冷板上。在玻璃小瓶中制备完所有样品后，打开真空泵，将样品瓶置于真空状态，然后取出真空源。使用压力表将所有样品置于真空下后，请仔细检查压力。如果小瓶泄漏，请确保盖紧紧拧紧，并且HSP和盖子衬垫的白色O形圈正确到位。将小瓶放入SPEU中以优化时间和温度，并以200 RPM搅拌。在70摄氏度下提取培养物一小时，并在37摄氏度下用粪便，污水，唾液和痰液样品提取稳定的同种型探测实验18小时。将冷板置于零下80摄氏度，以便在提取期完成后使用。提取完成后，将样品放在冷板上15分钟，以从HSP和小瓶顶部空间抽出水蒸气，然后将HSP转移到其套管中。在Entech软件上设置样品序列。打开程序，在仪器下拉菜单右侧的选项中选择 5800 和 序列。保存序列表，选择左侧的“运行”，如果空白 HSP 不是解吸器，则在“解吸器”中选择“从空白开始”。请注意，HSP将由SPR处理序列中的每个样品。让 SPR 热身。允许 SPR 自动运行所有样本。GC-MS端的序列将自动将数据记录在单独的文件中。通过选择“校准”，然后选择“外部标准化合物”下的“编辑化合物”、“名称和插入化合物”，将峰添加到处理方法中。添加化合物的名称、保留时间和量子信号目标离子。添加三个最大的峰，其中包括概率大于75%的化合物，确保化合物的每个识别离子的排列位于峰的中心。通过选择“确定”，然后选择“方法”和“保存”来保存它。设置过程方法后，继续定量和计算，然后查看和QEDIT定量结果以定量数据。在这里，真空辅助吸附剂提取之后，在GC-MS上进行热解吸，以调查细菌单培养物和共培养物的挥发性分布，并从人类粪便，唾液，污水和痰液样品中鉴定具有稳定同位素探测的活性挥发物。单一培养和共培养物包括细菌种类金黄色葡萄球菌、铜绿假单胞菌和鲍曼不动杆菌。在24小时和48小时的时间点从单培养物和共培养物中检测到43个注释的挥发性分子。与氘相比，13C掺入到完全标记的挥发性分子中更多。13C掺入2-丁酮，3-羟基，2，3-丁二酮二乙酰基，乙酸和苯酚中，用于所有粪便，污水和唾液样品。丙酮、丁酸和丙酸在唾液和污水中检测为标记。而二甲基三硫化物和二硫化物二甲基在粪便和唾液样品中均富集。挥发物1-丙醇、2-丁酮、二苯甲酮、乙醇和乙酸甲硫醇仅在污水中富集，2， 3-戊二酮在唾液中富集。氘被掺入来自唾液或污水样品的挥发物乙酸，苯甲醛，4-甲基二甲基三硫醚和苯酚中。培养的痰液样品中醋酸、二甲基三硫醚、丙酮和丙醇2-甲基比未培养的痰液样品更丰富。对变体的排列多变异分析PERMANOVA在囊性纤维化痰液样本挥发丰度的Bray-Curtis距离基质上进行。我们发现，捐赠样本的受试者解释了13C标记培养痰中51%的变异和未培养痰中33%的变异。在这里，显示了从七名囊性纤维化患者收集的培养痰液样本中挥发物中用13C葡萄糖进行稳定同位素标记的成功。图5A显示了大多数样品中具有较高13C掺入率的挥发物，大多数痰液样品中掺入率较低的13C百分比的挥发物显示在图5B中，少数痰液样品中转化率较低的分子显示在图5C中。约一分钟后，请务必仔细检查您的小瓶压力。破碎的真空会破坏VASE方法的灵敏度和速度。按照这一程序，如果进行了稳定同位素探测，则可以从剩余的材料中提取DNA，以鉴定可能有助于挥发性分子产生的微生物或物种。该方法也可用于检测任何样品类型的挥发物，而无需同位素探测。凭借灵敏度和低样品量的优点，许多应用都可以从该技术中受益。

Research

JoVE Journal

Biochemistry

Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy

模拟信号蛋白的功能：走向人工信号转导疗法

Signal transduction pathways, which control the response of cells to various environmental signals, are mediated by the function of signaling proteins ...

科研

生物化学

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

多聚-PAGE：生物样品中的用于拍摄方法和解决蛋白复合体

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

从人体二尖瓣提取蛋白质的优化方案

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

用生物层干涉法捕获小分子离子通道蛋白的相互作用动力学

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites 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 ...

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

使用顶空固相微萃取与气相色谱-质谱联用分析黑醋栗果实中的挥发性化合物

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars ...

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

毛细管电泳电喷雾电离质谱法绝对定量肌醇焦磷酸盐

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules ...

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

用完全纯化的组分重构Msp1提取活性

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

从 大麻 生物质中超声辅助提取大麻二酸

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center ...