这项工作由 NSF 植物基因组研究计划赠款 IOS-1238812 和生物学 IOS-1400818 博士后研究员资助。这项工作还由孟山都研究生奖学金给凯瑟琳 a. 萨蒙斯的资助。富布赖特非洲研究员学者计划 (2017-2018) 感谢授予 Nokwanda p 麻昆加的资金。我们非常感谢从杰西卡 Prenni 的 nanospray 来源和科罗拉多州立大学蛋白质组学和新陈代谢设施的捐赠。

1. 对 Nanospray 源的改质
<ol><li>使用改进的 nanospray 源进行叶喷雾 MS。由于叶喷雾 MS 不需要射流元件, 所以通过从源中移除 LC 探头来修改源。</li>
	<li>组装叶喷雾 MS 丝, 将应用电压的植物组织与适当的针插入源。将引脚焊接到绝缘导线的一端;将钳位焊在导线的另一端。 注: 夹子 (鳄鱼夹子类型) 可能或可能没有牙。对于小组织, 无牙钳是首选。一个可选的弹性臂与钳位可以添加到 nanospray 源, 以协助定位的植物组织。请注意, 本协议具体描述了如何在混合四极离子阱质量分析仪 ms 系统上执行叶喷雾 ms (见材料表);但是, 其他 MS 系统可能会被改变以执行这项技术6。耦合叶喷雾 MS 与便携式质谱仪实时化学分析可以在现场进行, 不需要运输植物材料到实验室19,20。</li>
	<li>将防静电地板垫放在电源下面的地板上, 以减少在使用高压时从电源上产生的电放电。</li>
</ol>2. 叶喷雾 ms 系统的研制
<ol><li>如果系统最近被使用, 允许它冷却到触摸和删除任何替代源和扫锥。附上 nanospray 叶喷雾 MS 源。</li>
	<li>创建具有适当电离参数设置的调谐文件, 如下所示: 鞘、辅助和扫气为 0;喷雾电压为 2-5 伏;毛细管温度到 150-250 °c;和 S 透镜 RF 水平到50。将调谐文件保存为所需的参数8、13。优化电压和温度的最佳电离的组织和化合物 (s) 的兴趣。 注: 良好的起始点为4伏和200°c。</li>
	<li>制作一个方法文件, 包括叶喷雾 ms 调谐文件: 正、负全 ms;7万的决议;1 x 106的 AGC 目标;最大值为200毫秒;和所需的扫描范围m/z。或者, 只使用1极性。</li>
</ol>3. 仪器、溶剂和植物组织的制备
注意:始终戴手套, 不要使用徒手处理过的植物组织。否则, 诸如聚乙二醇等污染物离子将主宰光谱。
<ol><li>将植物组织的分析带到与 MS 系统相同的房间, 以便快速取样。</li>
	<li>对于没有天然尖尖的植物组织, 在玻璃滑梯上使用刀片来切割锥形点 (图 2)。根据仪器灵敏度、组织类型和感兴趣的化合物 (例如, 长约5毫米的幼tortuosum叶) 确定分析所需的组织数量。<ol>
<li>tortuosum叶在10周后萌发成薄条, 各有一个锥形端形成一个点。</li>
	</ol>
</li>
	<li>使用镊子轻轻地选择将被夹紧的末端的植物组织。用镊子握住纸巾, 小心地将其转移到夹钳上。 注意: 如果电压为 on, 请勿触摸仪器电源。</li>
	<li>用夹钳调整挠性手臂和导线, 使组织与 ms 入口线保持一致, 这样植物组织与 ms 离子入口之间的距离为三重四极 (如TSQ) 和线性圈闭四极 (LTQ) 和10。-50 毫米离子阱质量分析仪 (例如, orbitrap)8。<ol>
<li>将导线的另一端插入源。如果第一次尝试产生低信号强度, 移动植物组织接近离子入口 (参考讨论为优选)。</li>
	</ol>
</li>
	<li>加载方法文件;命名数据文件并设置文件存储位置。然后通过单击 "播放" 打开 MS 系统, 然后单击 "开始" 开始获取数据。</li>
	<li>应用溶剂 (如甲醇), 使用吸管与凝胶加载尖端, 以最大的距离之间的手和高电压, 以保护用户。 注: 所需溶剂量取决于组织的大小、干燥度和质地, 一般为 2-20 µL tortuosum叶不需要添加任何溶剂。在电压不动的情况下, 小心地应用溶剂, 不要接触仪器电源。使用经酸洗的 LC-MS 级溶剂和玻璃器皿, 无洗涤剂。在某些组织中, 由于植物组织的自然含水量, 在不添加溶剂的情况下可以观察到信号。然而, 一个更大的信号强度和减少的 S/N 通常是通过应用溶剂的组织来实现。</li>
	<li>获取数据, 只要信号持续存在, 或者直到收集到足够的光谱, 通常为30六十年代。如果需要, 应用额外的溶剂保持较高的信号强度, 在较长的时间。停止数据获取并暂停 MS 系统。</li>
	<li>取下纸巾, 用100% 甲醇和无绒布擦拭钳洗。根据供应商的规格, 在大约 1-2 小时的采集后, 清洁 ms 离子入口。同时, 在不同组织类型的分析之间清除 MS 离子入口。</li>
</ol>4. 数据质量评估
<ol><li>打开数据文件, 目视检查基峰值质量 chronogram。检查信号强度是 ~ 1.0 x 107到 5.0 x 108。如果信号较低, 将组织移近离子入口。如果较高, MS 系统的前端将变得肮脏, 所以将组织进一步从离子入口。</li>
	<li>根据所产生的质谱中的感兴趣离子的存在或缺失, 改变参数。 注意: 协议可以在这里暂停。</li>
</ol>5. 串联质量碎片
<ol><li>确定哪些离子对串联质量碎片有兴趣 (ms/毫秒);1.0 x 105的质谱信号对于 ms/ms 的离子选择是足够的。</li>
	<li>制作一个新的方法文件, 其中包含的m/z的收录列表到4个小数位。点击全局列表和包含。在PRM 的属性下, 选择碎片能量 [例如, 30-50 的规范化碰撞能量是一个良好的起始范围] 和其他 ms/ms 参数。<ol>
<li>为了获得 mesembrine 生物碱的 ms/ms 数据, 片段的以下离子, 276.1583米/z, 290.1742米/z, 292.1897米/z, 在。 注意: ms 或 ms 数据采集可以在 ms 或以后的时间内立即执行。同样的组织可以在完整 ms 后保持钳位, 并且可以重用以获得 ms/ms 数据。但是, 如果 respraying 没有提供足够的信号, 请使用新的组织。</li>
	</ol>
</li>
	<li>加载 ms/毫秒方法文件和命名数据文件。打开 MS 系统, 开始获取数据, 必要时添加溶剂。当收集到足够的光谱, 通常在30六十年代之后, 停止获取。</li>
	<li>在分配片段离子时, 收集许多不同能量的碎片。 注: 由于叶喷雾 MS 缺乏色谱分离, 碎片谱可能含有许多离子, 而在不同能量下的分解将有助于使其清晰。</li>
</ol>6. 精确质量和串联质量碎片的假定识别
<ol><li>通过引用来自公共可用的代谢物数据库 (如 Metlin21、人类代谢物数据库22、大众银行23、脂质图24、美国国家研究所) 的精确质量测量来做出假定的标识。标准和技术 MS 搜索25, 尊重植物化学物质26, 或大亚湾27。</li>
	<li>由于这些数据库并非详尽无遗, 因此需要对具有化学特征的植物种类进行额外的文献综述。</li>
	<li>当 ms 或 ms 信息可用或文献时, 将碎片离子从叶喷雾 ms/ms 与上述数据库匹配。或者, 使用手动解释 ms/毫秒片段离子或由直接注入或 LC ms/ms 执行的真实标准的碎片。</li>
</ol>7. 数据分析
<ol><li>使用 msConvert 工具从 Proteowizard28将 MS 原始文件转换为 mzXML 文件。</li>
	<li>使用 R 中实现的 XCMS 软件包进行峰值拾取。采用直接输液处理方法对叶喷雾进行 MS 分析。 注意: 用于数据处理的注释好的脚本可以在 https://github.com/HegemanLab/Leaf-Spray-Code 中找到。</li>
	<li>为了获得半定量测量, 会计的实验变异性, 标准化的强度, 每一个代谢物的总离子电流 (TIC), 因为叶喷雾 MS 信号强度可能会有所不同, 部分原因是在定位的细微变化叶在来源和叶子的形状和大小的区别。</li>
	<li>或者, 使用供应商提供的软件进行数据分析或 MZmine2 (在 http://mzmine.github.io/中找到)29。</li>
</ol>

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作者没有什么可透露的。

该协议的成功使用依赖于植物种类、组织类型和目标化合物的各种步骤的优化。协议中描述的参数提供了一个良好的起点。以下实验决定需要做和测试: 是否使用 (1) 切开或未切割的组织和 (2) 溶剂或无溶剂, (3) 什么溶剂使用和在什么容量, (4) 从离子入口的组织距离应该是什么, (5) 电压振幅。优化的目标是找出产生连续信号的条件, 持续至少三十年代到几分钟。条件应提供足够的和可重现的信号强?...

植物含有多种化学性质的小分子。MS 是一种强有力的分析植物化合物的技术, 因为它能为代谢产物的检测和鉴定提供高灵敏度和特异性的元素成分1。最常见的是, ms 是在溶剂提取的样品, 这是分离的色谱之前, ms 分析1。然而, 使用液相色谱 (LC) 需要冗长的分析时间, 往往与广泛的样品准备1。相反, 直接化学分析的完整组织, 绕过色谱是一个非常迅速的技术, 需要最小的样品准备2。因此, 在色谱步骤可以被放弃的情况下, 直接的化学分析可以是非常有利的。
典型的 LC MS 为自然产品和新陈代谢研究依靠长的散装提取干燥或冷冻植物材料包含多组织和细胞类型3。或者, 直接化学分析, 如 MS 检测植物组织的代谢物, 可以隔离细胞类型和避免准备工件4。叶喷雾 ms, 也称为组织喷雾5,6, 是一个直接的环境电离 MS 技术, 这要求基本上没有样品准备5,7。叶喷雾 ms 与纸面喷雾 ms 密切相关, 这是一种具有电喷雾电离特性的环境电离技术, 可以检测沉积在纸7上的分析物。尽管名称, 叶喷雾 MS 是适用于各种类型的植物组织, 而不仅仅是叶子, 并已证明在水果, 种子, 根, 花卉组织, 块茎, 其中包括6,8,9, 10,11,12。该技术促进了内源植物化学物质直接从植物材料电离到质谱仪中检测8。叶喷雾 MS 还可以提供关于植物中不同组织类型的化学物质的空间分布的信息13。当叶喷雾 ms 与溶剂萃取和 LC ms 相比, 结果表明, 叶喷雾 ms 允许快速检测的表面代谢产物, 从独特的细胞类型, 如毛13。图 1说明了叶喷雾 MS 实验设置。直接电喷雾电离只在少量的源修改后发生。高压通过金属钳被应用到植物组织中, 产生一喷雾高电荷的水滴形成一个泰勒锥, 将离子传送到 MS 的离子入口. 电喷雾电离发生从植物的天然液体或溶剂李波爆炸到植物表面。一个尖尖的组织促进了电喷雾, 可以自然地发生或通过切割创建。
叶喷雾 MS 是一种快速的方法, 定性和半定量分析的完整的植物组织, 已发现效用广泛的应用。例如, 该技术被用来检测内源化合物, 以区分相关物种, 甚至评估在不同条件下生长的同一物种的变化。以前的研究通过测定紫珠中的代谢产物 (紫L) 表明了这种方法。12和美国人参(西洋参)6. 在后一种例子中, 可以在润湿生人参后发现人参皂甙、氨基酸和寡糖。野生和栽培西洋参被区分从块茎切片6。人参块茎的完整性保存后, 叶喷雾 MS, 允许随后的形态学和显微检查6。此外, 还可以检测植物样品中的外源化合物。在9水果和蔬菜的果皮或果肉上发现了一些杀虫剂 (脒、二苯胺、唑、linuron 和噻)。虽然这些研究和许多其他已经表明了叶喷雾 MS 的用途, 为各种特定目的, 一个详细的协议没有以前报告。
在这里, 协议描述不会集中于对特定组织或化合物的方法的优化。相反, Sceletium tortuosum (L.) n.e. (Aizoaceae) 中的 mesembrine 生物碱的检测被用作一个例子, 讨论在为物种、组织或化合物 (s) 第一次。tortuosum是南非半干旱 Karroo 地区特有的肉质植物。一种传统医学的 San 和同属的民族, 它被用于食欲和口渴抑制以及它的精神和镇痛作用14,15。目前, 标准提取物用于精神和神经心理疾病的治疗16,17。感兴趣的主要化合物包括生物碱 mesembrine 及其衍生物, 其中许多也见于相关的Sceletium物种15。tortuosum的野生和栽培种群都有 mesembrine 生物碱的浓度变化, 从而提出了质量控制挑战18。一种快速检测 mesembrine 生物碱的方法, 如叶喷雾 MS, 可用于监测Sceletium产品。由于以前没有对叶喷雾 MS 技术进行详细的可视化实验协议, 我们将用S. tortuosum的例子说明该方法, 并描述了以下内容: nanospray 源的修改、植物组织的选择和制备, 数据的获取, 结果的解释, 以及 MS 参数的优化。

<table><tbody><tr><td>Conn Pin</td><td>Digi-Key elctronics</td><td>WM2563CT-ND</td><td>pin will insert into Thermo Scientific source to provide voltage</td></tr><tr><td>small clamp</td><td>Digi-Key elctronics</td><td>314-1018-ND</td><td>CLIP MICRO ALLIGATOR COPPER 5A</td></tr><tr><td>large clamp</td><td>Digi-Key elctronics</td><td>290-1951-ND</td><td>ALLIGATOR CLIP NARROW NICKLE 5A</td></tr><tr><td>Heat shrink</td><td>Digi-Key elctronics</td><td>Q2Z1-KIT-ND</td><td>to cover soldering joints</td></tr><tr><td>NSI source Nanospray Ion Source</td><td>Thermo scientific</td><td>NA</td><td>Another brand will work if you are not using a Thermo instrument</td></tr><tr><td>Q Exactive- hybrid quadrupole Orbitrap</td><td>Thermo scientific</td><td>NA</td><td>Another brand will work if you are not using a Thermo instrument</td></tr><tr><td>Tune Software</td><td>Thermo scientific</td><td></td><td>Another brand will work if you are not using a Thermo instrument</td></tr><tr><td>Xcalibur Software</td><td>Thermo scientific</td><td></td><td></td></tr><tr><td>Plant of interest - &lt;em&gt;S. tortousum&lt;/em&gt;</td><td></td><td></td><td></td></tr></tbody></table>

leaf spray mass spectrometry: a rapid ambient ionization technique to directly assess metabolites from plant tissues

植物产生数以千计的小分子, 它们的化学性质多种多样。质谱 (MS) 是一种有效的分析植物代谢产物的技术, 因为它提供了高灵敏度和特异性的分子量。叶喷雾 MS 是一种环境电离技术, 植物组织用于直接化学分析通过电喷雾, 消除色谱的过程。这种取样代谢物的方法允许从完整的植物组织同时检测出多种化学类, 从而尽量减少所需的样品准备量。当使用高分辨率, 准确的质量 ms, 叶喷雾 ms 促进快速检测代谢物的兴趣。还可以用这种技术收集串联质量碎片数据, 以促进复合识别。准确的质量测量和碎片的结合有利于确认复合恒等式。叶喷雾 MS 技术只需要小的修改 nanospray 电离源, 是一个有用的工具, 进一步扩大的能力, 质谱仪。本文对南非传统药用植物Sceletium tortuosum (Aizoaceae) 的鲜叶组织进行了分析;用叶喷雾质谱检测了大量 mesembrine 生物碱。

在发芽后10周, 用叶喷雾质谱分析了新鲜收集的温室tortuosum叶。用叶喷雾 MS 检测tortuosum叶中代谢产物的实验工作流程如图 2所示。选择一叶, 切成带锥形端的薄条, 形成点, 并夹持叶状喷涂 MS 线钳装置。植物组织被安置了 ~ 30 毫米从离子入口并且与 x 和 y 轴一致。在tortuosum叶的叶喷雾剂启动后, 检测到没有任何溶剂的离子。Sceletiu...

Watch this Scientific Journal Video about Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues at JoVE.com

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

叶喷雾质谱法是一种直接的化学分析技术, 它能最大限度地减少样品的制备, 消除色谱, 从而快速检测植物组织中的小分子。

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 ...

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Research

JoVE Journal

Biochemistry

8.5K 次观看.  University of Minnesota. 叶喷雾质谱法是一种直接的化学分析技术, 它能最大限度地减少样品的制备, 消除色谱, 从而快速检测植物组织中的小分子。

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

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 ...

科研

化学

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

植物材料的快速高通量物种鉴定实时高分辨质谱法直接分析

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to the gas phase, and efficiently transferred to a detection system. Fizzy extraction takes advantage of the effervescence phenomenon. First, a carrier gas (here, carbon dioxide) is dissolved in the sample by applying overpressure and stirring the sample. Second, the sample chamber is decompressed abruptly. Decompression leads to the formation of numerous carrier gas bubbles in the sample liquid. These bubbles assist the release of the dissolved analyte species from the liquid to the gas phase. The released analytes are immediately transferred to the atmospheric pressure chemical ionization interface of a triple quadrupole mass spectrometer. The ionizable analyte species give rise to mass spectrometric signals in the time domain. Because the release of the analyte species occurs over short periods of time (a few seconds), the temporal signals have high amplitudes and high signal-to-noise ratios. The amplitudes and areas of the temporal peaks can then be correlated with concentrations of the analytes in the liquid samples subjected to fizzy extraction, which enables quantitative analysis. The advantages of fizzy extraction include: simplicity, speed, and limited use of chemicals (solvents).

Fizzy extraction is a new laboratory technique for analysis of volatile and semivolatile compounds. A carrier gas is dissolved in the liquid sample by applying overpressure and stirring the sample. The sample chamber is then decompressed. The analyte species are liberated to the gas phase due to effervescence.

挥发性有机化合物与大气压化学电离四极杆质谱联用

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

用于表征复杂天然有机物混合物的单吞吐量互补高分辨率分析技术

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

环境科学

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

生物化学

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a non-targeted approach, plant metabolites and xenobiotics of interest are identified and localized in plant tissues to uncover&#160;specific distribution patterns. Then, in silico prediction of potential metabolites (i.e., catabolites and conjugates) from the identified xenobiotics is performed. When a xenobiotic metabolite is located in the tissue, the type of enzyme involved in its alteration by the plant is recorded. These results were used to describe different types of biological reactions occurring in S. alba leaves in response to xenobiotic accumulation in the leaves. The metabolites were predicted in two generations, allowing the documentation of successive biological reactions to transform xenobiotics in the leaf tissues.

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

This method uses mass spectrometry imaging (MSI) to understand metabolic processes in S. alba leaves when exposed to xenobiotics. The method allows the spatial localization of compounds of interest and their predicted metabolites within specific, intact tissues.

通过质谱成像对 萨利克斯阿尔巴 叶中异种生物代谢的调查

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a ...

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 ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

常压LAESI质谱生物组织和生物膜的分子成像

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

生物学

使用 13C6-葡萄糖标记鉴定药用植物中的初级器官

13C6&#45;Glucose Labeling Associated with LC&#45;MS&#58; Identification of Plant Primary Organs in Secondary Metabolite Synthesis

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary metabolite in Parispolyphylla var. yunnanensis (Franch.) Hand. -Mzt. (PPY), Paris saponin (PS) has a variety of pharmacological activities and PPY is in increasing demand. This study established leaf, rhizome, and stem-vascular-bundle 13C6-Glucose feeding and non-feeding four treatments to precisely certify the primary organs involved in Paris saponins VII (PS VII) synthesis. By combining liquid chromatography-mass spectrometry (LC-MS), the 13C/12C ratios of leaf, rhizome, stem, and root in different treatments were quickly and accurately calculated, and four types of PS isotopic ion peak(M&#8722;) ratios were found: (M+1) &#8722;/M&#8722;, (M+2) &#8722;/M&#8722;, (M+3) &#8722;/M&#8722; and (M+4) &#8722;/M&#8722;. The results showed that the ratio of 13C/12C in the rhizomes of the stem-vascular-bundle and rhizome feeding treatments was significantly higher than that in the non-feeding treatment. Compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves increased significantly under leaf and stem-vascular-bundle feeding treatments. Simultaneously, compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves under rhizome treatment showed no significant difference. Furthermore, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the stem, root, and rhizome showed no differences among the four treatments. Compared to the non-feeding treatment, the ratio of the Paris saponin II (PS II) molecule (M+2) &#8722;/M&#8722; in leaves under leaf feeding treatment showed no significant difference, and the (M+3) &#8722;/M&#8722; ratio of PS II molecules in leaves under leaf feeding treatment were lower. The data confirmed that the primary organ for the synthesizing of PS VII is the leaves. It lays the foundation for future identification of the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants.

作者聚焦：使用 13C6-葡萄糖标记和 LC-MS 进行植物初级器官分析

The developed method of 13C6-Glucose labeling combined with liquid chromatography high-resolution mass spectrometry is versatile and lays the foundation for future studies on the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants, as well as the comprehensive utilization of these secondary metabolites.

13与LC-MS相关的C6-葡萄糖标记：次生代谢物合成中植物初级器官的鉴定

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary ...

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

摘要

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此视频中的章节

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）

植物材料的快速高通量物种鉴定实时高分辨质谱法直接分析

挥发性有机化合物与大气压化学电离四极杆质谱联用

用于表征复杂天然有机物混合物的单吞吐量互补高分辨率分析技术

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

通过质谱成像对萨利克斯阿尔巴叶中异种生物代谢的调查

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

常压LAESI质谱生物组织和生物膜的分子成像

¹³与LC-MS相关的C₆-葡萄糖标记：次生代谢物合成中植物初级器官的鉴定

¹³与LC-MS相关的C₆-葡萄糖标记：次生代谢物合成中植物初级器官的鉴定

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

摘要

探索更多视频

此视频中的章节

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）

植物材料的快速高通量物种鉴定实时高分辨质谱法直接分析

挥发性有机化合物与大气压化学电离四极杆质谱联用

用于表征复杂天然有机物混合物的单吞吐量互补高分辨率分析技术

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

通过质谱成像对 萨利克斯阿尔巴 叶中异种生物代谢的调查

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

常压LAESI质谱生物组织和生物膜的分子成像

13与LC-MS相关的C6-葡萄糖标记：次生代谢物合成中植物初级器官的鉴定

13与LC-MS相关的C6-葡萄糖标记：次生代谢物合成中植物初级器官的鉴定

通过质谱成像对萨利克斯阿尔巴叶中异种生物代谢的调查

¹³与LC-MS相关的C₆-葡萄糖标记：次生代谢物合成中植物初级器官的鉴定

¹³与LC-MS相关的C₆-葡萄糖标记：次生代谢物合成中植物初级器官的鉴定