| 基于UPLC－QTOF MS的鱼类鳃靶器官代谢组学分析方法研究 |
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| 基金项目:中央级公益性科研院所基本科研业务费专项（HSY202006M）；中国博士后科学基金面上项目（2020M681124）；黑龙江省博士后面上基金项目（LBH-Z20099） |
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| 中文摘要:针对鱼类鳃靶器官代谢组学研究缺乏准确高效的高通量检测技术的问题，建立了基于超高效液相色谱－飞行时间质谱联用技术（UPLC－QTOF MS）的分析方法。以图谱总峰面积、代谢物数目、相对峰面积的相对标准偏差（RSD）以及葡萄糖、L-亮氨酸、柠檬酸等6种代表性内源代谢物的响应值为指标，考察了不同实验条件对鳃组织中代谢物提取覆盖率、稳定性的影响。结果表明，采用加入样品－试剂比（mg∶μL）为1∶16的预冷甲醇－水（4∶1）低温研磨，并在-20 ℃下低温静置30 min后离心提取的方法时，鳃组织样本可获得最佳的提取效果。对数据采集过程中的色谱、质谱参数进行优化，进一步提高了代谢物的分离效果和信号强度，并利用质量控制（QC）样品进行了方法学考察。该方法具有较好的仪器精密度、方法精密度和样品稳定性，6种不同极性代表性代谢物的相对峰面积RSD均小于10%，可以满足鱼类鳃靶器官代谢组学研究的要求。研究结果为鱼类鳃靶器官的代谢组学研究提供了技术支撑。 |
| 中文关键词:代谢组学 样品前处理 鱼类 鳃 超高效液相色谱-飞行时间质谱 |
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| Research on Metabolomics Analysis Method for Fish Gills Target Organs Based on UPLC－QTOF MS |
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| Abstract:In view of the lack of accurate and efficient high-throughput detection techniques in metabolomics research of gill target organs in fish，an analysis method based on ultra-high performance liquid chromatography－quadrupole time-of-flight mass spectrometry（UPLC－QTOF MS） was established and optimized．In order to improve the extraction effect of metabolites in the sample，the total peak area，number of metabolites，relative standard deviations（RSDs） of relative peak area，and response values of glucose，L-leucine，citric acid，and other three representative endogenous metabolites with different species and polarities were used as indicators，and the effects of different experimental conditions on the extraction coverage and stability of metabolites in gill tissue were investigated．The results showed that the best extraction effect could be obtained by adding pre-cooled methanol－water（4∶1） at a sample－reagent ratio（mg∶μL） of 1∶16 to the gill tissue ground at low temperature，and holding for 30 min at －20 ℃，then centrifuged to extract gill tissue samples．Meanwhile，the chromatographic and mass spectrometry parameters in the data acquisition process were optimized，further improving the separation effect and signal intensity of metabolites．Methodological investigations were carried out by using quality control（QC） samples interspersed in the assay sequence．The results showed that this metabolomics analysis posessed good instrumental precision，method precision，and sample stability．The RSDs for the relative peak areas of the six representative metabolites with different polarities were lower than 10%，which meets the requirements for metabolomics research of fish gill target organs．The results provide an technical support for the metabolomic study of fish gill target organs． |
| Key Words:metabolomics sample pretreatment fish gill ultra-high performance liquid chromatography－quadrupole time-of-flight mass spectrometry（UPLC－QTOF MS） |
| 引用本文：刘英杰，姚明珠，李姗蔚，陈中祥，王鹏，孙言春.基于UPLC－QTOF MS的鱼类鳃靶器官代谢组学分析方法研究[J].分析测试学报,2022,41(8):1193-1199. |
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