我们承认美国国立卫生研究院资助P30ES013508和5T32GM008076的支持。我们也感谢Thermo Scientific的访问2.0和DRS筛。尤金Ciccimaro和马克·桑德斯Thermo Scientific的有益的讨论。

1。提取细胞样本<ol><li>对于10厘米板细胞:一个预先标注为10毫升的玻璃离心管中收集1.5毫升的解除在介质中的细胞悬浮液进入。对于贴壁线，细胞应被举起，轻轻刮在1.5ml保存在冰中的介质。 可选 :如果使用内部标准，添加一个适当的等分在这一步。 评论 :淬火细胞代谢的某些代谢产物是至关重要的。对于时间敏感的代谢产物分析，如冷甲醇提取的程序应该考虑17 </li><li>加入6毫升的氯仿（CHCl 3中）/甲醇（CH 3 OH）（2:1，体积/体积），以每10毫升含样品的玻璃试管。设置在摇床或低速多涡流混合器30分钟的样品。振摇后，将样品在1935×g离心10分钟，离心分离具有低的加速度/减速度设定在4℃以完全分离的相。 可选 </eM&gt;:要避免使用氯仿 ，拔牙可以进行二氯甲烷（CH 2 Cl 2中 ）。18,12 </li><li>使用长干巴斯德吸管，有机层（底部）转移到一个新的预标记的10毫升的玻璃离心管。继续到4.1。 </li><li>购买到干净的塑料2毫升的Eppendorf管中，将上层的水。在氮气中蒸发掉样品。继续2.6.2脱盐水相直接分析或继续到4.1。 </li></ol> 2。提取血清样本<ol><li> 20微升血清转移到一个塑料的2毫升的Eppendorf管中。 可选 :如果使用内部标准，在这一步中添加适当的等分。 </li><li>添加190微升CH 3 OH和涡持续10秒。 </li><li>添加380μl的氯仿和涡持续10秒。 </li><li>添加120微升H 2 O来诱导相分离。旋涡振荡的样品，持续10秒，并允许在室温下平衡10分。 </li><li>离心样品在8000×g离心10分钟，在4℃下以分离相。 </li><li>转移到一个干净的塑料2毫升的Eppendorf管较低的有机相。继续到4.1。 </li><li>购买到干净的塑料2毫升的Eppendorf管中，将上层的水。在氮气中蒸发掉样品。 </li><li>重悬样品200微升H 2 O也可以使用酸或碱试剂（0.1％乙酸，甲酸或0.1％氢氧化铵），优先提取pH敏感的代谢物。超声20分钟，冰重新完全暂停样品。 </li><li>激活C18离心柱用500μlCH 3 OH。 </li><li>平衡离心柱与500微升H 2 O洗涤两次，然后装入样品。如果使用酸/碱试剂在洗涤步骤中保持当前浓度。 </li><li>用500μlH 2 O冲洗脱盐样品。再次，如果酸/碱试剂，也用于保持当前浓度在洗涤步骤。 </li><li>成预先标记的干净的2毫升的Eppendorf管中，用两倍体积为200μl的80％CH 3 OH 中的H 2 O洗脱。再次，如果酸/碱试剂，也用于保持该浓度的洗涤步骤中。继续到4.1。 </li></ol> 3。从组织样本提取<ol><li>根据在纸巾上，使用冰冻组织10-100毫克之间。预先标记的2毫升低保留的Eppendorf管中，用1ml的PBS（1M，pH 6.8）中，在冰浴中添加整片的组织选择性的 :如果使用内部标准，将其添加到缓冲器中，然后加入组织。 </li><li>同质化的组织在冰用电动组织研磨30秒在每个Eppendorf公司。样品之间的，在两个单独的管中，CH 3 OH和H 2 O清洗组织研磨机。 </li><li>用塑料移液管的组织匀浆转移到一个预先标记的10毫升的玻璃离心管中。转让后，冲洗每个Eppendorf管CH 3 OH，并用1毫升2倍，添加洗涤液的玻璃试管中的组织匀浆。 </li><li>加入4毫升CHCl 3中，每个10ml玻璃管，管中含有的组织匀浆和CH 3 OH洗涤。设置在摇床或低速多涡流混合器30分钟的样品。 </li><li>振摇后，用低加速/减速设定在1935×g离心样品10分钟，以完全分离的相。 可选 :为了避免使用氯仿，提取已被开发，用CH 2 Cl 2中 。 </li><li>使用长干巴斯德吸管，有机层（底部）转移到一个新的预标记的10毫升的玻璃离心管。继续到4.1。 </li><li>使用长茎巴斯德移液管，将水层转移到一个新的预标记的10毫升的玻璃离心管中。 2.6.2脱盐或继续到4.1。 </li></ol> 4。 UPLC样品重新悬浮和过滤<ol><li>蒸发桑普氮气下的文件。 </li><li>复溶干燥样品的起始溶液，然后随意的的UPLC方法（ 见表1）在适当的音量。 50-100微升通常是一个理想的再悬浮量。振荡和/或向上和向下吸液管的样本，以帮助溶解分析物。 </li><li>将样品转移到0.22μm的尼龙管过滤器和旋转速度高达14,000 XG直到样品完全通过所述过滤器（约5分钟）。确保不存在任何可见的沉淀物样品中剩余。 </li><li>将上清液转移到一个预先标记的UPLC小瓶插入的滤波后的采样。盖小瓶，然后用手指滑动的小瓶，以消除任何气泡从底部样品瓶中。将样品放入冷冻自动进样器（4-5°C）。 </li></ol> 5。 UPLC设置<ol><li>使用液相色谱仪控制软件，创建一个运行基于有机样品中列出的条件<strONG&gt;表1。然后创建一个方法，在表1中列出的条件的水相关。 可选 :如果目标分析物的一组已知的是高息，优化的UPLC条件下使用这些化合物的重标记的模拟，并生成的方法，使用这些条件。 </li><li>让UPLC充分平衡。这应该包括一个完整的吸溶剂附加列前，按照制造商的一个新的列（通常为3-5倍柱体积）量平衡的方向。维护日志开始压力，以帮助诊断未来的问题。 </li></ol> 6。质谱仪设置<ol><li>使用控制软件，高分辨质谱，创建一个积极的模式使用在表2中列出的条件的方法。然后，在相同条件下，创建一个消极的方式方法。 可选 :如果一组已知的目标分析物的高整型erest，优化源和使用这些化合物的重标记的模拟中的光学条件下，生成的方法，使用这些条件。 </li><li>清洁和校准仪器，建立稳定的喷雾进入光谱仪，允许的时间校准解决方案，以充分消散从光源和光学系统。上可接受的稳定的喷雾，得到3-5％的相对标准偏差为作为LC方法被用来校准溶液以相同的流速注射强度超过至少100扫描。 </li><li>设置所有样本的序列，设置适当的注入量，运行的第一个采样前的空白。如果样品之间的结转或基质效应怀疑，运行足够的空白/洗涤。样品应被设置在一个随机的方式使用一个随机数发生器和一个键，以避免引入的任何批次的影响分析。 评论 :质量控制可能的目标包括稳定同位素标准或分析标准样品可极其宝贵的故障排除和保证可靠，重复性好，和有效的结果。只有一个标准样品，然后通过溶剂空白的一个技术上的复制可以用来访问到空白的信号强度和保留时间的偏差，以及交叉污染。 </li><li>开始顺序和压力波动，或亏损的信号强度在整个运行的问题，包括定期监测。如果检测到严重的问题，停止运行，重复6.2。 </li></ol> 7。微分分析<ol><li>两个工作流程都提出了这个协议。首先是通过筛2.0，赛默飞世尔出售的专有标签差分分析软件。第二个工作是通过差异分析开始之前，斯克里普斯研究所的一个免费平台，通过网上XCMS 11，手动检查的抽动或寻找任何已知的化合物样品中过滤色谱，以确保运行的重复性注塑/ UPLC /喷雾整个样品的稳定性。 </li><li>分子筛<ol><li>质谱仪筛安装在工作站的硬盘中的原始数据文件传输。 （ 注:运行筛与一个网络或USB端口连接的驱动器上存储的数据是不可取的，因为它可以限制分析速度。） </li><li>打开筛，并开始一个新的实验。 </li><li>进入筛，装入适当的文件。 </li><li>分配比较组，并选择一个单一的参考文件，以允许分子筛生成参数进行分析。 </li><li>按照提示按要求任何个人实验和调整任何参数。往往是最好开始用严格的参数，然后回去和放松的参数，更慷慨的设置可以延长分析时间。 </li><li>加载正确的加合物为积极或消极的模式列表中的参数。 </li></ol></li><li> XCMS在线<ol><li>转换文件线上XCMS到一个可以接受的格式。在我们的例子中，我们转换为mzXML的格式。19 </li><li>公开XCMS在线，并创建工作。 </li><li>上传和命名的数据集。只有两个数据集（ 如控制与治疗）可以上传仅限于单机XCMS头比较。 </li><li>从下拉列表中选择所需的设置。预填充的参数可用于不同的仪器设置，这些可以进一步修改后用于实验的需要。在我们的例子中，我们使用的HPLC-Orbi2设置。 </li><li>提交和确认工作。 </li></ol></li><li>数据分析<ol><li>列表中对于筛和XCMS的一个帧的功能，分别，分析完成后，将被填充。确保LC对齐覆盖任何地标峰。如果任何未对齐LC运行较大的偏差，具有里程碑意义的山峰，这可能表明样品有问题。 可选 :对于筛，如果使用内部标准，找到相应的峰组和规格化到选定的帧。对于在线XCMS，正常化可以在出口后的电子表格。 </li><li>绘制一个样人口（ 如控制样品）内的变异系数（CV）的数据。任何大的CV值可能指示一个或多个样品中的一个问题。想要的性状（ 如 p值&lt;0.05&gt; 1.5折变动，组内的低标准偏差等 ）的基础上对列表进行排序。 </li><li>检查适当的高峰，各组对齐，峰积分，高斯峰形，信号强度，同位素，加合物分配每个峰。 </li><li>将数据导出为需要作进一步的分析，或者生成有针对性的大规模名单进行确认，多级质谱实验。 </li></ol></li></ol>

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R., Neumann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAMERA:+An+Integrated+Strategy+for+Compound+Spectra+Extraction+and+Annotation+of+Liquid+Chromatography/Mass+Spectrometry+Data+Sets.">CAMERA: An Integrated Strategy for Compound Spectra Extraction and Annotation of Liquid Chromatography/Mass Spectrometry Data Sets.</a> Analytical Chemistry. 84 (1), 283-289 (2012).</li><li>Kanehisa, M., Goto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KEGG:+kyoto+encyclopedia+of+genes+and+genomes.">KEGG: kyoto encyclopedia of genes and genomes.</a> Nucleic Acids Res. 28 (1), 27-30 (2000).</li><li>Smith, C. A., O'Maille, G., 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=METLIN:+a+metabolite+mass+spectral+database.">METLIN: a metabolite mass spectral database.</a> Ther. Drug Monit. 27 (6), 747-751 (2005).</li><li>Wang, Y., Xiao, J., Suzek, T. O., Zhang, J., Wang, J., Bryant, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PubChem:+a+public+information+system+for+analyzing+bioactivities+of+small+molecules.">PubChem: a public information system for analyzing bioactivities of small molecules.</a> Nucleic Acids Res. 37 Web Server, W623-W633 (2009).</li><li>Wishart, D. S., Knox, C., 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=HMDB:+a+knowledgebase+for+the+human+metabolome.">HMDB: a knowledgebase for the human metabolome.</a> Nucleic Acids Res. 37 Database, D603-D610 (2009).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Canadian Journal of Biochemistry and Physiology. 37 (8), 911-917 (1959).</li><li>Folch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+isolation+and+purification+of+total+lipids+from+animal+tissues.">A simple method for the isolation and purification of total lipids from animal tissues.</a> J. Biol. Chem. 226, 497-509 (1957).</li><li>Avery, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+characterization+of+differential+ion+suppression+on+liquid+chromatography/atmospheric+pressure+ionization+mass+spectrometric+bioanalytical+methods.">Quantitative characterization of differential ion suppression on liquid chromatography/atmospheric pressure ionization mass spectrometric bioanalytical methods.</a> Rapid Communications in Mass Spectrometry. 17 (3), 197-201 (2003).</li></ol>

作者什么都没有透露。

调查内源性或外源性的生物转化，或捕捉利益的样本新陈代谢不相关的代谢组学研究提供了有力工具。输出的分辨率和灵敏度的技术用来分离和分析样品，产生的大数据集的处理能力，并有能力来挖掘有用的信息数据集（ 例如，精确的质量数据库检索）的技术尺度。最近，这已促成进步高分辨率质谱仪，高或超高效液相色谱法。差分分析软件分析瓶颈已经解决，并可以完成峰值检测调整保...

由于最近的技术进步，在人力资源管理领域的，不相关的，假设产生的代谢组学方法已经成为一种可行的方法复杂样品的分析。1质谱仪能够100,000决议，促进常规低的部分每百万（PPM）的质量精度，已成为广泛可从多个供应商2,3允许此质量准确度分析物的身份，同位素模式识别，加合物的鉴定中的初步分配更大的特异性和信心。4当加上适当的提取过程和高性能液相色谱或UPLC，复杂混合物可以分析来自于保留时间数据的附加 ​​特异性。5 UPLC色谱具有更大的效率，并允许更高的灵敏度，分辨率和分析时间，使由此产生的大的数据集可以被整合到任何一个更大的覆盖范围的代谢组可能的。6多个差分分析软件和有用的模式或个别分析物开采7,8,9,10,11推测点击可以使用峰值检测算法相结合，精确的质量为基础的化学式预测，预测碎片，初步确定化学数据库搜索。这种方法允许优先费时的完整的结构鉴定或更敏感和更具体的稳定同位素稀释的发展目标UPLC /选定或多个反应监测/ MS研究，是目前的黄金标准定量方法。12 生物样品的不同性质导致尿13，15日，14日 ，血清细胞或组织16提取协议的优化。此协议功能的细胞，血清和组织提取物。在适当的情况下，意见和附加引用已被列入修饰蒸发散的程序处理包括稳定同位素，包括特别是不稳定的代谢物。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Phosphate Buffered Saline</td><td>Mediatech</td><td>21-031-CM</td><td></td></tr><tr><td>Water (H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>W7-4</td><td>(optima)</td></tr><tr><td>Acetonitrile (CH&lt;sub&gt;3&lt;/sub&gt;CN)</td><td>Fisher Scientific</td><td>A996-4</td><td>(optima)</td></tr><tr><td>Methanol (CH&lt;sub&gt;3&lt;/sub&gt;OH)</td><td>Fisher Scientific</td><td>A454-4</td><td>(optima)</td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>A464-4</td><td>(optima)</td></tr><tr><td>Chloroform (CH&lt;sub&gt;3&lt;/sub&gt;Cl)</td><td>Sigma-Aldrich</td><td>366927</td><td>Hazard</td></tr><tr><td>Dichloromethane (CH&lt;sub&gt;2&lt;/sub&gt;Cl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Acros Organics</td><td>61030-1000</td><td>To replace chloroform</td></tr><tr><td>Diethyl Ether</td><td>Sigma-Aldrich</td><td>346136</td><td>To replace chloroform</td></tr><tr><td>Formic Acid (FA)</td><td>Fisher Scientific</td><td></td><td>(optima)</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;OH</td><td>Fisher Scientific</td><td>A470-250</td><td>(optima)</td></tr><tr><td>Ammonium formate (HCOONH&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>78314</td><td></td></tr><tr><td>MicroSpin C18 Columns</td><td>Nest Group Inc</td><td>SS18V</td><td></td></tr><tr><td>Pasteur Pipettes</td><td>Fisher Scientific</td><td>13-678-200</td><td></td></tr><tr><td>10 ml Glass Centrifuge Tubes</td><td>Kimble Chase</td><td>73785-10</td><td></td></tr><tr><td>10 ml Plastic Centrifuge Tubes</td><td>CellTreat</td><td>CLS-4301-015</td><td></td></tr><tr><td>LC Vials (glass)</td><td>Waters</td><td>60000751CV</td><td></td></tr><tr><td>LC Inserts (glass)</td><td>Waters</td><td>WAT094171</td><td></td></tr><tr><td>LC Vials (plastic)</td><td>Waters</td><td>186002640</td><td></td></tr><tr><td>0.22 &amp;mu;m Filters</td><td>Corning</td><td>8169</td><td>nylon</td></tr><tr><td>2 ml Eppendorf Tubes</td><td>BioExpress</td><td>C-3229-1</td><td>Low Retention</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>High Resolution Mass Spectrometer</td><td>Thermo Scientific</td><td>LTQ XL-Orbitrap</td><td></td></tr><tr><td>HPLC/UPLC</td><td>Waters</td><td>nanoACQUITY UPLC</td><td></td></tr><tr><td>Source</td><td>Michrom</td><td>Thermo Advance Source</td><td></td></tr><tr><td>Differential Analysis Software</td><td>Thermo Scientific</td><td>SIEVE 2.0</td><td></td></tr><tr><td>nanoACQUITY C18 BEH130</td><td>Waters</td><td>186003546</td><td>1.7 &amp;mu;m particle size, 150 mm x 100 &amp;mu;m</td></tr><tr><td>Acentis Express C8</td><td>Sigma-Aldrich</td><td>54262</td><td>2.7 &amp;mu;m particle size, 15 cm x 200 &amp;mu;m</td></tr></tbody></table>

untargeted metabolomics from biological sources using ultraperformance liquid chromatography-high resolution mass spectrometry (uplc-hrms)

在这里，我们提出了一个工作流程，分析代谢公司的利益，包括生物样品，细胞，血清，或组织。先将样品分离成极性和非极性组分的液 - 液固相萃取，部分纯化的，以方便下游分析。这两种水溶液（极性代谢物）和有机相（非极性代谢物）的初始提取处理，调查广泛的代谢物。代谢物分开由不同的液相色谱方法，根据其分区属性。在该方法中，我们提出了微流的超高性能（UP）的液相色谱方法，但该协议是可扩展的，更高的流量和较低的压力下。介绍进入质谱仪可以通过优化源条件一般或复合。在正面和负面的模式在全扫描模式进行了广泛的离子检测m / z范围点多面广，使用高分辨率最近Ç仪器alibrated。出版商无差分析进行生物信息学平台。这种方法的应用包括代谢途径筛选，生物标志物的发现和药物开发。

结果显示选定的数据从6小时治疗的SH-SY5Y胶质母细胞瘤的农药和细胞与线粒体复合物I抑制剂鱼藤酮。为简单起见，只有有机相正模式数据。对样品进行处理，并如上所述分析（ 图1，表1，表2）和无标签定量，分子筛和XCMS的在线到两个差分分析平台上加载。虽然有大量的点击率（ 图2，图3），这些功能包括用于差分分析两种方案的确定可能出现的文物，集成不善的山峰，?...

Watch this Scientific Journal Video about Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS at JoVE.com

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

不相关的代谢组学研究提供了一个假说，产生一种代谢轮廓的快照。此协议将展示从细胞，血清或组织中的代谢产物的提取和分析。代谢物甲范围进行调查，采用液 - 液固相萃取，微流超高效液相色谱/高分辨率质谱（UPLC-HRMS）耦合到差分分析软件。

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 ...

untargeted-metabolomics-from-biological-sources-using

Research

JoVE Journal

Chemistry

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

22.3K 次观看.  University of Pennsylvania. 不相关的代谢组学研究提供了一个假说，产生一种代谢轮廓的快照。此协议将展示从细胞，血清或组织中的代谢产物的提取和分析。代谢物甲范围进行调查，采用液 - 液固相萃取，微流超高效液相色谱/高分辨率质谱（UPLC-HRMS）耦合到差分分析软件。

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

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

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

生物化学

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

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

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 ...

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes.&#160;Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 &#181;l. &#160;Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.

The reliability of results in metabolomics experiments depends on the effectiveness and reproducibility of the sample preparation. Described is a rigorous and in-depth method that enables extraction of metabolites from biological fluids with the option of subsequently analyzing up to thousands of compounds, or just the compound classes of interest.

多步制备技术，以恢复多个代谢化合物的种类进行深入和信息化代谢组学分析

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital ...

生物工程

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 ...

Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss of muscle mass). Plasma samples were taken at the beginning of the intervention and after 30 days of consuming the functional meals. A semi-targeted ultra-high-performance chromatography coupled with tandem mass (UPLC-MS/MS) analysis was carried out to identify phenolic compounds and their metabolites. Plasma proteins were precipitated with ethanol and the samples were concentrated and resuspended in the mobile phase (1:1 acetonitrile: water) before injection into the UPLC-MS/MS instrument. Separation was carried out with a C18 reverse-phase column, and compounds were identified using their experimental mass, isotopic distribution, and fragment pattern. Compounds of interest were compared to those of data banks and the internal semi-targeted library. Preliminary results showed that the major metabolites identified after the intervention were phenylacetic acid, glycitin, 3-hydroxyphenylvaleric acid, and gomisin M2.

The goal of this protocol is to detect phenolic metabolites in plasma using a semi-targeted chromatography-mass spectrometry method.

老年人血浆中酚类代谢物的半靶向超高效色谱耦合质谱分析

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss ...

An Integrated Workflow of Identification and Quantification on FDR Control-Based Untargeted Metabolome

Untargeted metabolomics techniques are being widely used in recent years. However, the rapidly increasing throughput and number of samples create an enormous amount of spectra, setting challenges for quality control of the mass spectrometry spectra. To reduce the false positives, false discovery rate (FDR) quality control is necessary. Recently, we developed a software for FDR control of untargeted metabolome identification that is based on a Target-Decoy strategy named XY-Meta. Here, we demonstrated a complete analysis pipeline that integrates XY-Meta and metaX together. This protocol shows how to use XY-meta to generate a decoy database from an existing reference database and perform FDR control using the Target-Decoy strategy for large-scale metabolome identification on an open-access dataset. The differential analysis and metabolites annotation were performed after running metaX for metabolites peaks detection and quantitation. In order to help more researchers, we also developed a user-friendly cloud-based analysis platform for these analyses, without the need for bioinformatics skills or any computer languages.

We constructed an untargeted metabolomic workflow that integrated XY-Meta and metaX together. In this protocol, we displayed how to use XY-Meta to generate a decoy spectral library from open access spectra reference, and then performed FDR control and used the metaX to quantitate the metabolites after identifying the metabolomics spectra.

基于FDR对照的非靶向代谢组的鉴定和定量集成工作流程

Untargeted metabolomics techniques are being widely used in recent years. However, the rapidly increasing throughput and number of samples create an ...

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography ...

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 ...

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）

摘要

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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）

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

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

多步制备技术，以恢复多个代谢化合物的种类进行深入和信息化代谢组学分析

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

老年人血浆中酚类代谢物的半靶向超高效色谱耦合质谱分析

基于FDR对照的非靶向代谢组的鉴定和定量集成工作流程

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

敏感，大规模定量代谢组学的策略

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）

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）

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

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

多步制备技术，以恢复多个代谢化合物的种类进行深入和信息化代谢组学分析

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

老年人血浆中酚类代谢物的半靶向超高效色谱耦合质谱分析

基于FDR对照的非靶向代谢组的鉴定和定量集成工作流程

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

敏感，大规模定量代谢组学的策略

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