Name: untargeted liquid chromatography-mass spectrometry-based metabolomics analysis of wheat grain
Uploaded: 2020-03-13T12:00:00
Description: Watch this Scientific Journal Video about Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain at JoVE.com

作者感谢西澳大利亚州总理的农业和食品奖学金计划（西澳大利亚州政府就业、旅游、科学和创新系）和总理研究员西蒙·库克教授（该中心数字农业、柯廷大学和默多克大学）。现场试验和谷物样品收集得到了西澳大利亚州政府区域版税计划的支持。我们感谢格兰特利·施泰纳和罗伯特·法兰西对现场审判的贡献。NCRIS资助的澳大利亚生物平台公司因设备融资而得到认可。

此方法适用于 30 个样本（每个样本约 150 个种子）。这里使用了10个不同田间种植小麦品种的三个生物复制。
1. 谷物制备
<ol><li>从-80°C储存中检索样品（全谷物）。 注：如果从多个季节采集样品，建议在收获后不久冷冻干燥种子。这样可以最大限度地减少不同存储期后可能出现的代谢物浓度变化。为此，将种子转移到15 mL塑料离心管（大约300个种子将填充管），并盖上铝?...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+LC-MSE+Lipidomic+Analysis+using+a+Shotgun+Approach+and+Its+Application+to+Biomarker+Detection+and+Identification+in+Osteoarthritis+Patients.">Comprehensive LC-MSE Lipidomic Analysis using a Shotgun Approach and Its Application to Biomarker Detection and Identification in Osteoarthritis Patients.</a> Journal of Proteome Research. 9 (5), 2377-2389 (2010).</li><li>Sangster, T., Major, H., Plumb, R., Wilson, A. J., Wilson, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pragmatic+and+readily+implemented+quality+control+strategy+for+HPLC-MS+and+GC-MS-based+metabonomic+analysis.">A pragmatic and readily implemented quality control strategy for HPLC-MS and GC-MS-based metabonomic analysis.</a> Analyst. 131 (10), 1075-1078 (2006).</li><li>Dunn, W. 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A., Solomon, 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=The+necrotrophic+effector+SnToxA+induces+the+synthesis+of+a+novel+phytoalexin+in+wheat.">The necrotrophic effector SnToxA induces the synthesis of a novel phytoalexin in wheat.</a> New Phytologist. 200 (1), 185-200 (2013).</li><li>Bowne, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drought+Responses+of+Leaf+Tissues+from+Wheat+Cultivars+of+Differing+Drought+Tolerance+at+the+Metabolite+Level.">Drought Responses of Leaf Tissues from Wheat Cultivars of Differing Drought Tolerance at the Metabolite Level.</a> Molecular Plant. 5 (2), 418-429 (2012).</li><li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sharing+and+community+curation+of+mass+spectrometry+data+with+Global+Natural+Products+Social+Molecular+Networking.">Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking.</a> Nature Biotechnology. 34 (8), 828-837 (2016).</li><li>Shahaf, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+WEIZMASS+spectral+library+for+high-confidence+metabolite+identification.">The WEIZMASS spectral library for high-confidence metabolite identification.</a> Nature Communications. 7 (1), 12423 (2016).</li></ol>

在这里，我们提出了一种基于LC-MS的无靶向代谢组学方法，用于小麦颗粒分析。该方法通过将第三个移动相引入反向相梯度，将四种采集模式（反向相位和脂质抗逆相与正负电离）合并为两种模式。该组合方法每电离极性产生约500个生物学相关特征，其中大约一半在小麦品种的强度上明显不同。不同小麦品种颗粒代谢物浓度的显著变化表明生物化学的变化，这可能与抗病性、耐应力性和其他对谷?...

了解农业中基因、环境和管理实践之间的相互作用，可以更准确地预测和管理产品产量和质量。植物代谢物受基因组、环境（气候、降雨等）等因素的影响，在农业环境中，作物的管理方式（即肥料、杀菌剂等的应用）都受其影响。与基因组不同，甲目瘤受所有这些因素的影响，因此代谢组学数据在特定时间提供这些相互作用的生化指纹。基于代谢组学的研究通常有两个目标之一：第一，加深对生物体的生物化学的理解，并帮助解释与生理学相关的扰动（非生物或生物应激）的反应机制;第二，将生物标志物与正在研究中的扰动联系起来。在这两种情况下，拥有这种知识的结果都是一种更精确的管理策略，以实现提高产量和质量的目标。
据预测，植物美塔博洛米含有数千种具有不同物理化学性质的小分子。目前，没有代谢组学平台（主要是质谱和核磁共振光谱学）可以在单个分析中捕获整个甲胺。开发此类技术（样品制备、代谢物提取和分析），在单个分析运行中尽可能大地覆盖甲胺，是代谢学研究人员的主要目标。以前对小麦颗粒的无目标代谢组学分析将多个色谱分离和采集极性的数据和/或仪器相结合，以扩大甲目覆盖。然而，这需要为每个方式单独编制和获取样品。例如，Beleggia等人2为GC-MS的极性分析物分析，以及对非极性分析物的GC-MS分析，编制了一个派生样本。Das等人3使用GC和LC-MS方法提高分析覆盖率;然而，这种方法通常需要上述单独的样品准备以及两个独立的分析平台。以前使用GC-MS22、3、43,4和LC-MS33、55平台对小麦颗粒的分析为GC-MS产生了50至412（55个已识别）特征，409个用于GC-MS和LC-MS的组合，几千个用于LC-MS脂质分析5。通过将至少两种模式合并为一种分析，可以保持扩展的美表体覆盖率，从而增加生物解释的丰富性，同时节省时间和成本。
为了允许通过反向相色谱法有效地分离各种脂质，现代脂质学方法在洗脱溶剂6中通常使用高比例的异丙醇，为可能通过色谱学而无法解决的脂质类提供方便。为了进行有效的脂质分离，起始移动相在有机成分7中也比典型的反向相色谱方法高得多，后者考虑其他类别的分子。梯度开始时的高有机成分使得这些方法不太适合许多其他类分子。最值得注意的是，反向相液色谱法采用二元溶剂梯度，从大部分水性成分开始，随着色谱的洗脱强度增加，有机含量增加。为此，我们力求将两种方法结合起来，在单个分析中实现代谢物的脂质和非脂类的分离。
在这里，我们提出了一种使用第三个移动相的色谱法，并使用单个样品制备和一个分析柱实现结合传统的反向相和脂质组学的色谱法。我们采用了许多质量控制措施和数据过滤步骤，这些措施以前在主要临床代谢组学研究中实施过。这些方法有助于确定具有高技术可重复性和生物相关性的强健特性，并排除不符合这些标准的特征。例如，我们描述对池质样本8、QC校正9、数据过滤99、1010和缺失特征11的归记分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>13C6-sorbitol</td>
			<td>Merck Sigma-Aldrich</td>
			<td>605514</td>
			<td></td>
		</tr>
		<tr>
			<td>2-aminoanthracene</td>
			<td>Merck Sigma-Aldrich</td>
			<td>A38800-1 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>ThermoFisher Scientific</td>
			<td>FSBA955-4</td>
			<td>Optima LC-MS grade</td>
		</tr>
		<tr>
			<td>Ammonium formate</td>
			<td>Merck Sigma-Aldrich</td>
			<td>516961-100 mL</td>
			<td>&#62;99.995%</td>
		</tr>
		<tr>
			<td>Analyst TF</td>
			<td>Sciex</td>
			<td></td>
			<td>Version 1.7</td>
		</tr>
		<tr>
			<td>AnalyzerPro software</td>
			<td>SpectralWorks Ltd.</td>
			<td></td>
			<td>Data processing software used for step 7.2. Version 5.7</td>
		</tr>
		<tr>
			<td>AnalyzerPro XD sortware</td>
			<td>SpectralWorks Ltd.</td>
			<td></td>
			<td>Data processing software used for step 7.5. Version 1.4</td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Sartorius. Precision Balances Pty. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>d6-transcinnamic acid</td>
			<td>Isotec</td>
			<td>513962-250 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Ajax Finechem Pty. Ltd.</td>
			<td>A2471-500 mL</td>
			<td>99%</td>
		</tr>
		<tr>
			<td>Freeze dryer (Freezone 2.5 Plus)</td>
			<td>Labconco</td>
			<td>7670031</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Schott bottles (100 mL, 500 mL, 1 L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vials (2 mL) and screw cap lids (pre-slit)</td>
			<td>Velocity Scientific Solutions</td>
			<td>VSS-913 (vials), VSS-SC91191 (lids)</td>
			<td></td>
		</tr>
		<tr>
			<td>Installation kit for Sciex TripleToF</td>
			<td>Sciex</td>
			<td>p/n 4456736</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>ThermoFisher Scientific</td>
			<td>FSBA464-4</td>
			<td>Optima LC-MS grade</td>
		</tr>
		<tr>
			<td>Laboratory blender</td>
			<td>Waring commercial</td>
			<td></td>
			<td>Model HGBTWTS3</td>
		</tr>
		<tr>
			<td>Leucine-enkephalin</td>
			<td>Waters</td>
			<td>p/n 700008842</td>
			<td>Tuning solution</td>
		</tr>
		<tr>
			<td>Metaboanalyst</td>
			<td><a href="https://www.metaboanalyst.ca/MetaboAnalyst/faces/home.xhtml" target="_blank">https://www.metaboanalyst.ca/MetaboAnalyst/faces/home.xhtml</a></td>
			<td></td>
			<td>Web-based analytical pipeline for high-throughput metabolomics. Free, web-based tool. Version 4.0.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>ThermoFisher Scientific</td>
			<td>FSBA456-4</td>
			<td>Optima LC-MS grade</td>
		</tr>
		<tr>
			<td>Miconazole</td>
			<td>Merck Sigma-Aldrich</td>
			<td>M3512-1 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge (Eppendorf 5415R)</td>
			<td>Eppendorf (Distributed by Crown Scientific Pty. Ltd.)</td>
			<td></td>
			<td>5426 No. 0021716</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (2 mL)</td>
			<td>SSIbio</td>
			<td>1310-S0</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Office Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peak View software</td>
			<td>Sciex</td>
			<td></td>
			<td>Version 1.2 (64-bit)</td>
		</tr>
		<tr>
			<td>Pipette tips (200 uL, 100 uL)</td>
			<td>ThermoFisher Scientific</td>
			<td>MBP2069-05-HR (200 uL), MBP2179-05-HR (1000 uL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes (200 uL, 1000 uL)</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic centrifuge tubes (15 mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>NUN339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Progenesis QI</td>
			<td>Nonlinear Dynamics</td>
			<td></td>
			<td>Samll molecule discovery analysis software. Version 2.3 (64-bit)</td>
		</tr>
		<tr>
			<td>Sciex 5600 triple ToF mass spectrometer</td>
			<td>Sciex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Screw-cap lysis tubes (2 mL) with ceramic beads</td>
			<td>Bertin Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium formate</td>
			<td>Merck Sigma-Aldrich</td>
			<td>456020-25 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue lyser/homogeniser</td>
			<td>Bertin Technologies</td>
			<td></td>
			<td>Serial 0001620</td>
		</tr>
		<tr>
			<td>Volumetric flasks (10 mL, 50 mL, 100 mL, 200 mL, 1 L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>IKA Works Inc. (Distributed by Crown Scientific Pty. Ltd.)</td>
			<td></td>
			<td>001722</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>ThermoFisher Scientific</td>
			<td>FSBW6-4</td>
			<td>Optima LC-MS grade</td>
		</tr>
		<tr>
			<td>Water&#39;s Acquity LC system equipped with quaternary pumps</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water&#39;s Aquity UPLC 100mm HSST3 C18 column</td>
			<td>Waters</td>
			<td>p/n 186005614</td>
			<td></td>
		</tr>
	</tbody>
</table>

untargeted liquid chromatography-mass spectrometry-based metabolomics analysis of wheat grain

了解基因、环境和农业实践中的管理之间的相互作用，可以更准确地预测和管理产品产量和质量。代谢组学数据提供了在给定时间时刻读取这些相互作用的信息，并且对生物体的生化状态信息。此外，代谢物的单个代谢物或面板可用作产量和质量预测和管理的精确生物标志物。据预测，植物美塔博洛米将包含数千种具有不同物理化学特性的小分子，为生物化学洞察生理特性和生物标志物发现提供了机会。为了利用这一点，代谢学研究人员的一个关键目标是在单个分析中捕获尽可能多的物理化学多样性。在这里，我们提出了一种基于液相色谱-质谱的非靶向代谢组学方法，用于分析田间生长的小麦颗粒。该方法使用液体色谱四元溶剂管理器引入第三个移动相，并将传统的反向相梯度与脂质梯度相结合。详细介绍了谷物制备、代谢物提取、工具分析和数据处理工作流程。观察了良好的质量精度和信号可重复性，该方法每电离模式产生约500个生物学相关特征。此外，还确定了小麦品种之间的代谢物和脂质特征信号显著不同。

植物美谱受其基因组和环境的组合影响，此外，在农业环境中，作物管理系统。我们证明，小麦品种之间的遗传差异可以在代谢物水平上观察到，在这里，500多种测量化合物表明，仅在谷物中品种之间的浓度就明显不同。对于负电化和正电化模式，观察到内部标准（下图2）的良好质量精度（<10 ppm 误差）和信号重现性（<20% RSD）。Table 3?...

Watch this Scientific Journal Video about Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain at JoVE.com

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

提出了一种对小麦粒代谢物和脂质进行非靶向分析的方法。该协议包括醋酸代谢物提取方法和反向相液色谱-质谱方法，在正负电喷雾电相成像模式下采集。

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 ...

代谢物受基因组环境和管理实践等因素影响。了解这些因素之间的相互作用可以更准确地预测和管理产品的产量和质量。通过结合第三个移动相，而不是传统的两个，该技术允许分离和检测更广泛的代谢物。代谢组学可应用于生物学的任何领域。首先，对生物体的生物化学有更深入的了解，帮助解释它们对与生理学有关的非生物或生物应激的反应。其次，将生物标志物与研究的扰动性联系在一起。由于异丙醇是一种粘性溶剂，因此应在低耀斑速率下引入，在将组合物增加至 98% 之前应使用足够的平衡时间。要开始制备谷物，请使用实验室搅拌机研磨谷物。高速运行搅拌机 20 秒，然后重复。从底座上拆下搅拌机罐。点击搅拌杯的侧面，将任何粗糙的颗粒带到样品表面。粗糙的颗粒可以丢弃或存储。将细磨颗粒从搅拌机转移到两毫升塑料微型离心管。在萃取的同一天，准备萃取溶剂，如手稿中所述。接下来，将200毫克细磨颗粒放入两毫升微型离心管中。在管中的颗粒中加入500微升萃取溶剂。此外，通过向空管中加入 500 微升萃取溶剂来准备空白。使用均质器，设置为 6，500 RPM，混合溶剂和颗粒 20 秒。然后，再重复20秒。然后，在摄氏四度和16，100倍G下将管离心5分钟。将上一液转移到两毫升塑料管中，并保留颗粒。在颗粒上重复两次提取过程。将三种超自然剂混合，产生约1.5毫升的总萃取量，通过涡流混合。要对用于质量控制的所有谷物提取物进行一个集合样本，请将每个谷物提取物的 55 微升转移到两毫升管和涡流中。然后，将该池样品的 50 微升等分转移到玻璃瓶中。将每个颗粒样品提取物的 50 微升等分转移到玻璃瓶中。如书面手稿所述，准备液相色谱质谱法所需的解决方案。在 LCMS 分析的当天，准备 100 毫升 5%乙酰丙酸，每毫升亮氨酸素含有 200 毫微克。将 950 微升溶液添加到含有 50 微升等分的谷物提取物的玻璃小瓶中。通过涡流混合小瓶中的内容物。首先，按照手稿和制造商的用户指南，设置校准仪器。接下来，使用LCMS级溶剂（包括移动相和洗涤溶剂）清洗和冲洗LC流体系统。使用 LC 方法启动条件平衡 LC 系统，确保柱压稳定。设置仪器序列表。在样品序列的开头注射甲酸钠以检查仪器校准。首先分析溶剂和制备空白，然后对系统调节进行集中质量控制样品，然后进行随机抽样列表。随着技术复制的定期运行质量控制样本。在序列的末尾运行两个质量控制样本。在序列运行时，检查数据质量，包括内部标准质量精度和信号可重复性。要检查信号的可重复性，对叠加光谱的目视检查就足够了。用于准备谷物提取物的解决方案中包括了四个内部标准。在LCMS期间，对正负电离模式均观察到了良好的质量精度和内部标准的单一可重复性。根据对空白的分析，判断负模中的421个信号和正模中的835个信号是伪影。这些信号以等于或大于粒样品中平均单强度 5% 的强度的空白出现。删除伪影和进一步筛选数据后，负模式返回 483 个功能，正模式返回 523 个功能。大约250个特征与生物学有关，而且小麦品种的强度差异很大。质量控制措施（如包含准备空白、内部标准和集合样本）在此协议中非常重要。

Research

JoVE Journal

Biochemistry

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

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

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 ...

科研

生物化学

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 ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

通过反相液相-质谱法对无细胞蛋白合成代谢的绝对定量

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

通过基因表达分析的改良方法从谷物种子获取高质量的转录数据

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

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 ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

肽聚糖的液相色谱质谱和生物信息学半定量分析

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

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

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem 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

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

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

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 ...