School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
Phenome Centre Birmingham, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
J Proteome Res. 2021 Jan 1;20(1):831-840. doi: 10.1021/acs.jproteome.0c00660. Epub 2020 Nov 25.
Metabolic phenotyping of tissues uses metabolomics and lipidomics to measure the relative polar and nonpolar (lipid) metabolite levels in biological samples. This approach aims to understand disease biochemistry and identify biochemical markers of disease. Sample preparation methods must be reproducible, sensitive (high metabolite and lipid yield), and ideally rapid. We evaluated three biphasic methods for polar and nonpolar compound extraction (chloroform/methanol/water, dichloromethane/methanol/water, and methyl tert-butyl ether [MTBE]/methanol/water), a monophasic method for polar compound extraction (acetonitrile/methanol/water), and a monophasic method for nonpolar compound extraction (isopropanol/water). All methods were applied to mammalian heart, kidney, and liver tissues. Polar extracts were analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) and nonpolar extracts by C reversed-phase UHPLC-MS. Method reproducibility and yield were assessed using multiple annotated endogenous compounds (putatively and MS/MS annotated). Monophasic methods had the highest yield and high reproducibility for both polar (positive ion: median relative standard deviation (RSD) < 18%; negative ion: median RSD < 28%) and nonpolar (positive and negative ion: median RSD < 15%) extractions for heart, kidneys, and liver. The polar monophasic method extracted higher levels of lipid than biphasic polar extractions, and these lipids caused minimal detection suppression for other compounds during HILIC UHPLC-MS. The nonpolar monophasic method had similar or greater detection responses of all detected lipid classes compared to biphasic methods (including increased phosphatidylinositol, phosphatidylserine, and cardiolipin responses). Monophasic methods are quicker and simpler than biphasic methods and are therefore most suited for future automation.
组织代谢组学研究使用代谢组学和脂质组学来测量生物样本中相对极性和非极性（脂质）代谢物的水平。这种方法旨在了解疾病的生物化学，并确定疾病的生化标志物。样品制备方法必须具有重现性、灵敏度（高代谢物和脂质产率），并且理想情况下快速。我们评估了三种用于提取极性和非极性化合物的两相方法（氯仿/甲醇/水、二氯甲烷/甲醇/水和甲基叔丁基醚[MTBE]/甲醇/水）、一种用于提取极性化合物的单相方法（乙腈/甲醇/水）和一种用于提取非极性化合物的单相方法（异丙醇/水）。所有方法均应用于哺乳动物心脏、肾脏和肝脏组织。极性提取物通过亲水相互作用色谱（HILIC）超高效液相色谱-质谱（UHPLC-MS）进行分析，非极性提取物通过 C 反相 UHPLC-MS 进行分析。使用多个注释的内源性化合物（推定和 MS/MS 注释）评估了方法的重现性和产率。单相方法对于极性（正离子：中位数相对标准偏差（RSD）<18%；负离子：中位数 RSD<28%）和非极性（正离子和负离子：中位数 RSD<15%）提取物均具有最高的产率和高重现性心脏、肾脏和肝脏。极性单相方法提取的脂质水平高于两相极性提取物，并且在 HILIC UHPLC-MS 中，这些脂质对其他化合物的检测抑制最小。非极性单相方法与两相方法相比，具有更高的所有检测到的脂质类别的检测响应（包括增加的磷脂酰肌醇、磷脂酰丝氨酸和心磷脂响应）。单相方法比两相方法更快、更简单，因此最适合未来的自动化。
