代谢组学样本预处理方法

样品预处理策略的选择有助于给定实验的成功，因为它影响了观察到的代谢物谱和数据的质量。给定研究中样品制备方法的能力和局限性会影响生物学解释的准确性。

Pre-treatment of metabolomics samples must meet the following conditions: 1) non-selective; 2) few and rapid steps; 3) reproducible; 4) include a metabolic termination process. Currently, a single pre-treatment does not meet all of these requirements.

从生物标志物发现的角度来看，最矛盾的需求之一是代谢终止，包括来自细胞，植物和组织的样品。代谢终止的目的是使用低温，酸或快速加热来停止代谢过程。但是，代谢过程非常快，并且通常的时间尺度小于1 s，例如ATP，6-磷酸葡萄糖和腺苷。在适当的时间范围内实施适当的终止步骤可能非常困难，并且添加终止步骤可能会导致某些代谢物的无意下降或损失。

For stable metabolites, metabolic termination is of little significance, but for unstable metabolites that are easily degraded or converted, the step of metabolic termination is important. Although the proportion of unstable metabolites is low, it is also possible that these low proportions of metabolites are precisely the important biomarkers. Therefore, determining which metabolites are not affected by different termination/storage conditions has an important role in the maturation of the field of metabolomics and biomarker discovery.

Different metabolomics studies have different requirements for sample pre-processing. In discovery-oriented非靶向代谢组学研究，在分析之前，生物样品经历越少的预处理越少，以便在样品中检测到尽可能多的代谢产物。因此，研究人员通常使用非选择性的预处理方法，例如具有有机溶剂（例如血清或血浆样品预处理），样品稀释（例如尿液样品预处理）的蛋白质沉淀。在有针对性的元bolomics studies, sample pretreatment is followed by liquid-liquid extraction or solid-phase extraction to remove matrix interferences after protein precipitation with organic solvents for further separation and enrichment of target metabolites. These operations greatly improve the sensitivity and dynamic range of the assay.

Metabolomics workflow (Alseekhet al., 2021).

生物流体样品的预处理方法

• Direct dilution injection

通常将其应用于尿液样品，并使用纯净水的典型稀释比为1:10倍。

• 溶剂沉淀

The addition of organic solvents (methanol, acetonitrile, ethanol, acetone or a combination thereof) to biological fluid samples such as serum, plasma, etc. can remove proteins and disrupt the cross-linking between proteins and metabolites, and the resulting metabolite concentration is representative of the total metabolite concentration. The best metabolite coverage is obtained with acetone/methanol mixtures and methanol precipitation.

• Ultrafiltration

超滤是制备和释放生物样品的常见方法，这些方法是根据分析物的分子质量分离的。例如，3000 DA膜可以将小分子质量代谢物与蛋白质或其他大分子分开。与溶剂沉淀相比，超滤对极性分子有偏见，并导致疏水代谢物物质的显着损失。

• 固相提取（SPE）

SPE广泛用于靶向生物分析，步骤如下：1。分析物在溶剂表面吸附；2.通过浸湿去除分析物吸附能力弱的干扰物；3.分析物使用溶剂洗脱。目前，C18和聚苯乙烯 - 二苯基吸附剂组合已用于非靶向相位代谢组学研究。

• 固相微萃取（SPME）

在vivo sampling avoids the metabolic profile changes caused by the oxygen, solvent, and pH conditions to which the sample may be exposed during sampling, and the corresponding activation of various biological processes. The SPEM technique provides a good balance between hydrophilic and hydrophobic metabolite extraction. This technique can be applied to the in vivo sampling of circulating blood metabolites in mice.

• 湍流色谱（TFC）

The TFC method allows direct injection of untreated serum into the LC-MS. TFC meets the conditions of large particles (25-50um) and small particle size (0.5-1.0um), where large molecules cannot be retained and small molecules can be detected. the potential advantages of TFC are high volume, high automation and minimal sample handling, which can reduce the introduction of foreign contaminants in the sample handling process and Unintentional sample loss.

基于质谱的代谢组学的样品预测技术（Raterinket al.，2014年）

References

1. Alseekh，S.，Aharoni，A.，Brotman，Y.，et al。(2021). Mass spectrometry-based metabolomics: A guide for annotation, quantification and best reporting practices. Nature methods, 18(7), 747-756.
2. Raterink, R. J., Lindenburg, P. W.,et al。（2014）。基于质谱的代谢组学的样品预测技术的最新发展。分析化学的TRAC趋势，61，157-167。

相关部分

Metabolomics Service

血液和血浆代谢组学服务

尿液代谢组学服务

撕裂代谢组学Service