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利用非靶标代谢组的方法测定土壤可提取有机碳组分   

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摘要:土壤有机碳组分,特别是能被微生物利用的可溶性有机碳组分,对于理解土壤有机碳的动态循环及微生物的代谢活性至关重要。土壤可溶性有机碳组分主要由植物和微生物来源的代谢物和裂解细胞组成,它是一个复杂的代谢物库。非靶标代谢组学分析方法在生物医学领域已被广泛用于分析复杂提取物中的代谢组分。气相色谱质谱联用和液相色谱质谱联用都可以用来分析这些小分子代谢组分,但是气相色谱质谱联用只能分析具有挥发性的物质,在代谢物的检测上具有一定的局限性。因此,本实验主要介绍利用液相色谱质谱联用分析非靶标代谢组分的方法来分析土壤可溶性有机碳组分。

关键词: 土壤可溶性有机碳组分, 非靶标代谢组, 液相色谱质谱联用

材料与试剂

  1. 2 ml离心管
  2. 小钢珠 (直径5 mm)
  3. 0.3 mg/ml二氯苯丙氨酸溶液 (分析纯)
  4. 甲醇溶液 (分析纯)
  5. 无菌水
  6. 液相色谱进样小瓶
  7. 枪头 (100 μl, 1 ml)

仪器设备

  1. 涡旋仪
  2. 制冰机
  3. 离心机
  4. 超声仪
  5. 超高相液相色谱 (AB SCIEX,BEH C18柱)
  6. 质谱仪 (AB SCIEX,ABSCIEX-Triple TOF 5600)
  7. 移液枪

实验步骤

  1. 称取500 mg 过2 mm筛的鲜土于2 ml的离心管中。
  2. 加入1颗直径5 mm的小钢珠,20 μl的0.3 mg/ml的二氯苯丙氨酸,500 μl的甲醇溶液 (甲醇和水体积比4:1)。
  3. 涡旋混匀离心管中的样品和溶液,在冰上超声萃取10 min。
  4. 重复冰上超声萃取2次。
  5. 在-20 °C下静置30 min。
  6. 3220 x g,4 °C条件下离心15 min取上清液于进样小瓶中。
  7. 提取的上清液在超高效液相色谱串联飞行时间质谱UPLC-TripleTOF系统下进行测定。
    注:如果不是立刻测定,提取的上清液可以放在-20 °C条件下保存。
    关于如何提取土壤中的有机碳并在液相色谱质谱仪上进行上机测定的方法是参考 Swenson等人(2015,2019)发表的关于利用非靶标代谢组分的方法分析土壤中的可提取有机碳组分方法探究。

结果与分析

将下机获取的原始wiff/.scan文件,通过GNPS网站上的batch converting方法(https://ccms-ucsd.github.io/GNPSDocumentation/fileconversion/) 转换为mzXML格式的文件。转换格式后的mzXML文件就可以导入Mzmine2 (http://mzmine.github.io/) 软件中,提取同时包含一级质谱分子离子峰 (MS1) 和二级质谱分子结构 (MS2) 的代谢组分特征表和mgf格式的质谱信息文件。将这两个文件导入全球自然产物分子网络(Global Natural Products Social Molecμlar Networking, GNPS, https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp) 中,在线构建分子网络来判断小分子的种类。
        通过Mzmine2软件提取代谢组信息数据的具体步骤如下:        
        Raw data methods ---- import file,导入mzXML文件
        Raw data methods ---- peak detection ---- mass detection (scan MS level:1)
        Raw data methods ---- peak detection ---- mass detection (scan MS level:2)
        Raw data methods ---- peak detection ---- chromatogram builder
        Peak list methods ---- peak detection ---- chromatogram deconvolution
        Peak list methods ---- isotopes ---- isotopic peak grouper
        Peak list methods ---- filtering ---- peak list row filter
        Peak list methods ---- gap filtering ---- peak finder
        Peak list methods ---- export -----export to csv file
        Peak list method ---- export for / submit to GNPS
        注:每一步中参数的调整依据数据的情况进行相应的设置。

致谢

本实验得到国家自然科学基金项目 (31870480) 的资助。

参考文献

  1. Swenson, T. L., Jenkins, S., Bowen, B. P. and Northen, T. R. (2015). Untargeted soil metabolomics methods for analysis of extractable organic matter. Soil Biol Biochem 80: 189-198.
  2. Swenson, T. L. and Northen, T. R. (2019). Untargeted soil metabolomics using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Methods Mol Biol 1859: 97-109.
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:张考萍, 褚海燕. (2021). 利用非靶标代谢组的方法测定土壤可提取有机碳组分. Bio-101: e2003679. DOI: 10.21769/BioProtoc.2003679.
How to cite: Zhang, K. P. and Chu, H. Y.  (2021). Untargeted Metabolomics Method for the Analysis of Molecμlar Composition of Soil Dissolved Organic Carbon. Bio-101: e2003679. DOI: 10.21769/BioProtoc.2003679.
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