大鼠脑组织被提取物的高分辨核磁共振代谢组学分析
Summary
The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).
Abstract
Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.
Introduction
小鼠模型已被广泛利用在大脑研究1。基因型-表型的相关性进行了研究在小鼠和大鼠的大脑通过研究基因表达在一方面的RNA和/或蛋白水平，和形态，功能，电和/或行为的表现型上的其它2-6。然而，为了完全理解联的表型基因型的机制，就必须进行调查的分子事件的下游蛋白的表达， 即 。生化基板在其酶作用7的代谢。这要求领导，在过去10至15年，以代谢研究在生物学8,9的许多分支的复兴。虽然古典代谢的研究往往集中在特定途径的细节，新的代谢途径是面向正在考虑组织的全球性代谢谱的全方位调查。这个概念的一个后果是用于分析工具，最大限度地减少对特定的代谢途径和/或类型的化合物的偏压明显需要。然而，传统的生化测定是基于需要进行化验之前被指定的特定分析物的特定的化学反应。其中每一个与此相反，如核磁共振（NMR）光谱法和质谱（MS）（ⅰ）的光谱技术是基于生化化合物的特定分子（物理）性能产生了一个或若干个不同信号中的频谱在一个实验的过程中检测到的;以及（ii）检测大量每个实验的不同化合物。
因此，每一个光谱包含代谢物的整个范围的综合信息。出于这个原因，光谱方法可用于代谢组适当的工具，如不事先选择需要进行关于分析物的性质进行测定8 </suP>。其结果是，这些技术自然地借给自己探索性的研究，因为它们极大地方便了意想不到的代谢变化的检测。
虽然NMR光谱法和质谱可以互换使用，用于许多代谢物的分析中，每个方法具有特定的优点和缺点，最近已审查10。简言之，核磁共振光谱，通常可以从粗提取物中进行，且不需要样品化合物的分析前的色谱分离。相比之下，MS可与气体或液体色谱（GC或LC）分离，除去最近特别发展，如质谱成像。在一些特殊情况下，如糖 的分析，液相色谱分离可能成为一种必然的核磁共振光谱为好，因为不同的糖的共振线质子显著重叠（1H）NMR谱。然而，1 H NMR光谱没有字符omatographic分离仍然是最流行的，几乎普遍应用代谢组学核磁共振方法。通常，样品制备是更费时和复杂的MS比为核磁共振光谱。由于基体效应的严重问题是许多在NMR谱，其中它们可以导致相当衰减的信号不太常见的比在MS。代谢物的定量可以用任何一种方法来实现。然而，需要为MS多种标准化合物由于变化的代谢物之间的基体效应和离子化效率。与此相反，每个样品只有一个标准是需要的NMR波谱分析，因为在适当的测量条件下，后者的方法在本质上是定量由于通过所观察到的细胞核的严格线性的核磁共振响应。 NMR的主要缺点是其相对低的灵敏度。 MS，尤其是LC-MS，是由数个数量级比核磁共振更敏感;因为这个原因，MS要优于核磁共振对分析在非常低的浓度存在的化合物。另一方面，在NMR实验的无损性质是通过MS具有明显的优势;以这种方式，NMR可以反复对同一样品， 例如进行，不同的NMR活性核，例如1 H，磷-31（31 P），碳-13（13 C），氟-19（19 F）的等 ，如无材料以NMR消耗相对于MS的测量。
既NMR和MS可以在不同的模式下被使用，每一个是最适合于特定化学特性的化合物的检测。例如，31 P NMR往往更适合于1 H-NMR进行的适度浓缩的磷酸化化合物的分析，虽然几乎所有的磷酸化代谢物还含有质子。然而，它们的1 H NMR信号可以通过从其它的非磷酸化的化合物的1 H NMR信号被遮蔽，而后者则显然不会造成在31 P NMR谱的背景信号。在模拟的情况下，19 F NMR分析是优选的氟化合物， 如氟化的药物（从内源性代谢物没有背景信号），而13 C-NMR的特例是感兴趣的几乎全部，如果13 C-命运标记的外源性代谢前体需要遵循，由于13 C同位素（ 约 1％）的极低的天然丰度。许多质谱仪在任负离子模式或正离子模式下工作。因此，重要的是要知道前面的分析是否被观察的离子是带负电或带正电的。我们在此关注的协议对脑组织代谢的通过1 H和31 P NMR光谱法的分析，因为这种方法会产生在（ⅰ）需要的样品制备时间方面有大量的重要代谢物浓度以低成本ND所需的代谢物的定量（ⅱ）的努力。所有实验中可以使用一个标准的湿化学实验室的设备和高分辨率核磁共振光谱设施来进行。而且要求在下面的协议部分进行了描述。
Protocol
Representative Results
Discussion
NMR光谱法是用于测量溶液中的化学化合物的浓度在很重现的和准确的方式的一种有效方法。然而，为了获得高质量的数据，必须坚持有关样品制备和分析的某些规则。中代谢物浓度的NMR谱的测定，既不产生也不NMR信号的接收占主导地位的定量误差，除非观察到的信号的强度接近的检测阈值（特别是弱信号）。在所有其他情况下的生物变异性，样品制备技术（提取效率，化合物的物理和化学稳定性…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Support by Centre National de la Recherche Scientifique (CNRS, UMR 6612 and 7339) is gratefully acknowledged.
Materials
| Isoflurane | Virbac | Vetflurane | Anesthetic for animals |
| Isoflurane vaporizer | Ohmeda | Isotec 3 | Newer model available: Isotec 4 |
| Scalpel, scissors, forceps, clamps | Harvard Apparatus Fisher Scientific |
various various |
Surgical equipment for animals |
| Freeze-clamp tool | homebuilt | n/a | Tong with aluminium plates, to be inserted in liquid nitrogen for cooling |
| Dewar | Nalgene | 4150-4000 | |
| Liquid nitrogen | Air Liquide | n/a | |
| Nitrogen gas | Air Liquide | n/a | |
| Nitrogen evaporator | Organomation Associates | N-EVAP 111 | Can be replaced by homebuilt device |
| Mortar | Sigma-Aldrich | Z247472 | |
| Pestle | Sigma-Aldrich | Z247510 | |
| Tissue homogenizer | Kinematica | Polytron | With test tubes fitting homogenizer shaft |
| Electronic scale | Sartorius | n/a | |
| Methanol | Sigma-Aldrich | M3641 | |
| Chloroform | Sigma-Aldrich | 366910 | |
| Glass centrifuge tubes | Kimble | 45500-15, 45500-30 | Kimax 15-mL, 30-mL tube |
| Microcentrifuge tubes | Kimble | 45150-2 | Kimax 2-mL tube; should replace "Eppen-dorf" tube if compatible with centrifuge rotor |
| polystyrene pipettes | Costar Corning | Stripettes | 5 and 10-mL volumes |
| Deuterochloroform | Sigma-Aldrich | 431915 | 99.96 % deuterated |
| Deuterium oxide | Sigma-Aldrich | 423459 | 99.96 % deuterated |
| Deuterium chloride | Alpha Aesar | 42406 | 20 % in deuterium oxide |
| Sodium deuteroxide | Sigma-Aldrich | 164488 | 30 % in deuterium oxide |
| Lyophilizer | Christ | Alpha 1-2 | |
| Cold centrifuge | Heraeus | Megafuge 16R | |
| pH meter | Eutech Cybernetics | Cyberscan | |
| CDTA | Sigma-Aldrich | D0922 | |
| Cesium hydroxide | Sigma-Aldrich | 516988 | |
| NMR tubes | Wilmad | 528-PP | |
| NMR stem coaxial insert | Sigma-Aldrich | Z278513 | By Wilmad |
| NMR pipettes | Sigma-Aldrich | Z255688 | |
| Pipettes | Eppendorf | Research | With tips for volumes from 0.5 to 1000 μL |
| Pipet-Aid | Drummond | XP | |
| NMR spectrometer | Bruker | AVANCE 400 | including probe and other accessories |
| NMR software | Bruker | TopSpin 1.3 | newer version available: Topspin 3.2 |
| Water-soluble standard compounds | Sigma-Aldrich | various | |
| Phospholipid standard compounds | Avanti Polar Lipids Doosan Serdary Sigma-Aldrich |
various various various |
Source for plasmalogens, but may be < 70 – 80 % purity |
| Methylenediphosphonate | Sigma-Aldrich | M9508 | |
| TSP-d4 | Sigma-Aldrich | 269913 |
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