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 border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Mediatech</td>
			<td>21-031-CM</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water (H2O)</td>
			<td>Fisher Scientific</td>
			<td>W7-4</td>
			<td>(optima)</td>
		</tr>
		<tr>
			<td>Acetonitrile (CH3CN)</td>
			<td>Fisher Scientific</td>
			<td>A996-4</td>
			<td>(optima)</td>
		</tr>
		<tr>
			<td>Methanol (CH3OH)</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 (CH3Cl)</td>
			<td>Sigma-Aldrich</td>
			<td>366927</td>
			<td>Hazard</td>
		</tr>
		<tr>
			<td>Dichloromethane (CH2Cl2)</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>&nbsp;</td>
			<td>(optima)</td>
		</tr>
		<tr>
			<td>NH4OH</td>
			<td>Fisher Scientific</td>
			<td>A470-250</td>
			<td>(optima)</td>
		</tr>
		<tr>
			<td>Ammonium formate (HCOONH4)</td>
			<td>Sigma-Aldrich</td>
			<td>78314</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MicroSpin C18 Columns</td>
			<td>Nest Group Inc</td>
			<td>SS18V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pasteur Pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 ml Glass Centrifuge Tubes</td>
			<td>Kimble Chase</td>
			<td>73785-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 ml Plastic Centrifuge Tubes</td>
			<td>CellTreat</td>
			<td>CLS-4301-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LC Vials (glass)</td>
			<td>Waters</td>
			<td>60000751CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LC Inserts (glass)</td>
			<td>Waters</td>
			<td>WAT094171</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LC Vials (plastic)</td>
			<td>Waters</td>
			<td>186002640</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.22 &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>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>High Resolution Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>LTQ XL-Orbitrap</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HPLC/UPLC</td>
			<td>Waters</td>
			<td>nanoACQUITY UPLC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Source</td>
			<td>Michrom</td>
			<td>Thermo Advance Source</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Differential Analysis Software</td>
			<td>Thermo Scientific</td>
			<td>SIEVE 2.0</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>nanoACQUITY C18 BEH130</td>
			<td>Waters</td>
			<td>186003546</td>
			<td>1.7 &mu;m particle size, 150 mm x 100 &mu;m</td>
		</tr>
		<tr>
			<td>Acentis Express C8</td>
			<td>Sigma-Aldrich</td>
			<td>54262</td>
			<td>2.7 &mu;m particle size, 15 cm x 200 &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

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

使用氢交换质谱分析蛋白质动力学

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

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, ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

利用二次 Nanoelectrospray 电离耦合高分辨质谱的实时呼吸分析

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

活蛙 (爪蟾) 胚胎单细胞代谢的微探针毛细管电泳质谱

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

利用毛细管区电泳串联质谱技术进行大尺度自上而下蛋白质组学

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

Imaging Corrosion at the Metal-Paint Interface Using Time-of-Flight Secondary Ion Mass Spectrometry

Corrosion developed at the paint and aluminum (Al) metal-paint interface of an aluminum alloy is analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS), illustrating that SIMS is a suitable technique to study the chemical distribution at a metal-paint interface. The painted Al alloy coupons are immersed in a salt solution or exposed to air only. SIMS provides chemical mapping and 2D molecular imaging of the interface, allowing direct visualization of the morphology of the corrosion products formed at the metal-paint interface and mapping of the chemical after corrosion occurs. The experimental procedure of this method is presented to provide technical details to facilitate similar research and highlight pitfalls that may be encountered during such experiments.

Time-of-flight secondary ion mass spectrometry is applied to demonstrate the chemical mapping and corrosion morphology at the metal-paint interface of an aluminum alloy after being exposed to a salt solution compared with a specimen exposed to air.

利用飞行时二次离子质谱法成像金属颜料界面的腐蚀

Corrosion developed at the paint and aluminum (Al) metal-paint interface of an aluminum alloy is analyzed using time-of-flight secondary ion mass ...

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.

使用高分辨率X射线吸收光谱在低温下制备生物样品以进行形态分析

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...

3D Depth Profile Reconstruction of Segregated Impurities Using Secondary Ion Mass Spectrometry

The presented protocol combines excellent detection limits (1 ppm to 1 ppb) using secondary ion mass spectrometry (SIMS) with reasonable spatial resolution (~1 &#181;m). Furthermore, it describes how to obtain realistic three-dimensional (3D) distributions of segregated impurities/dopants in solid state materials. Direct 3D depth profile reconstruction is often difficult to achieve due to SIMS-related measurement artifacts. Presented here is a method to identify and solve this challenge. Three major issues are discussed, including the i) nonuniformity of the detector being compensated by flat-field correction; ii) vacuum background contribution (parasitic oxygen counts from residual gases present in the analysis chamber) being estimated and subtracted; and iii)&#160;performance of all steps within a stable timespan of the primary ion source. Wet chemical etching is used to reveal the position and types of dislocation in a material, then the SIMS result is superimposed on images obtained via scanning electron microscopy (SEM). Thus, the position of agglomerated impurities can be related to the position of certain defects. The method is fast and does not require sophisticated sample preparation stage; however, it requires a high-quality, stable ion source, and the entire measurement must be performed quickly to avoid deterioration of the primary beam parameters.

The presented method describes how to identify and solve measurement artifacts related to secondary ion mass spectrometry as well as obtain realistic 3D distributions of impurities/dopants in solid state materials.

使用二次离子质谱法重建分离杂质的 3D 深度剖面

The presented protocol combines excellent detection limits (1 ppm to 1 ppb) using secondary ion mass spectrometry (SIMS) with reasonable spatial ...

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described&#8212;single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)&#8212;to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

Post-transcriptional modifications of RNA represent an understudied layer of translation regulation that has recently been linked to central nervous system plasticity. Here, sample preparation and liquid chromatography-tandem mass spectrometry approach is described for simultaneous characterization of numerous RNA modifications in single neurons.

使用质谱法表征单个神经元中的RNA修饰

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...