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）
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1。提取细胞样本对于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 …
Representative Results
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，?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Reagent | |||
| Phosphate Buffered Saline | Mediatech | 21-031-CM | |
| Water (H2O) | Fisher Scientific | W7-4 | (optima) |
| Acetonitrile (CH3CN) | Fisher Scientific | A996-4 | (optima) |
| Methanol (CH3OH) | Fisher Scientific | A454-4 | (optima) |
| Isopropanol | Fisher Scientific | A464-4 | (optima) |
| Chloroform (CH3Cl) | Sigma-Aldrich | 366927 | Hazard |
| Dichloromethane (CH2Cl2) | Acros Organics | 61030-1000 | To replace chloroform |
| Diethyl Ether | Sigma-Aldrich | 346136 | To replace chloroform |
| Formic Acid (FA) | Fisher Scientific | (optima) | |
| NH4OH | Fisher Scientific | A470-250 | (optima) |
| Ammonium formate (HCOONH4) | Sigma-Aldrich | 78314 | |
| MicroSpin C18 Columns | Nest Group Inc | SS18V | |
| Pasteur Pipettes | Fisher Scientific | 13-678-200 | |
| 10 ml Glass Centrifuge Tubes | Kimble Chase | 73785-10 | |
| 10 ml Plastic Centrifuge Tubes | CellTreat | CLS-4301-015 | |
| LC Vials (glass) | Waters | 60000751CV | |
| LC Inserts (glass) | Waters | WAT094171 | |
| LC Vials (plastic) | Waters | 186002640 | |
| 0.22 μm Filters | Corning | 8169 | nylon |
| 2 ml Eppendorf Tubes | BioExpress | C-3229-1 | Low Retention |
| Equipment | |||
| High Resolution Mass Spectrometer | Thermo Scientific | LTQ XL-Orbitrap | |
| HPLC/UPLC | Waters | nanoACQUITY UPLC | |
| Source | Michrom | Thermo Advance Source | |
| Differential Analysis Software | Thermo Scientific | SIEVE 2.0 | |
| nanoACQUITY C18 BEH130 | Waters | 186003546 | 1.7 μm particle size, 150 mm x 100 μm |
| Acentis Express C8 | Sigma-Aldrich | 54262 | 2.7 μm particle size, 15 cm x 200 μm |
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Cite This Article
Snyder, N. W., Khezam, M., Mesaros, C. A., Worth, A., Blair, I. A. Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS). J. Vis. Exp. (75), e50433, doi:10.3791/50433 (2013).