通过LC-MS / MS分析离体和体内稳定同位素标记的中心碳代谢和相关途径。,Nature Protocols

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通过LC-MS / MS分析离体和体内稳定同位素标记的中心碳代谢和相关途径。
Nature Protocols ( IF 13.1 ) Pub Date : 2019-02-01 , DOI: 10.1038/s41596-018-0102-x
Min Yuan _{1,

2} , Daniel M Kremer _{3,

4} , He Huang _{1,

2,

5} , Susanne B Breitkopf _{1,

2,

5} , Issam Ben-Sahra ₆ , Brendan D Manning ₇ , Costas A Lyssiotis _{3,

8,

9} , John M Asara _{1,

2,

5}

Affiliation

靶向串联质谱（LC-MS / MS）在分析从生物来源（例如细胞，体液和组织）中提取的小分子时非常有用。在这里，我们提出了一个协议，用于分析非放射性稳定同位素碳13（13C）和氮15（15N）在中央碳代谢和相关途径中的极性代谢物中的掺入。我们的平台利用带有极性切换功能的选定反应监测（SRM）和酰胺亲水相互作用液相色谱（HILIC），使用混合三重四极杆（QQQ）质谱仪捕获碳和氮掺入选定代谢物中的跃迁。该方案代表了先前发布的针对未标记物种的目标代谢组学方案的扩展，已被广泛用于在各种生物学环境中追踪营养物质的代谢，例如13C标记的葡萄糖，13C-谷氨酰胺和15N-谷氨酰胺（例如，细胞培养实验和通过ip注射进行体内小鼠标记）。对SRM信号进行积分，以产生每种标记形式的峰面积阵列，这些峰面积用作进一步分析的输出。然后将处理后的数据用于获得目标分子标记的程度和分布（称为通量组学）。每种方法都可以基于已知的未标记Q1 / Q3 SRM过渡进行定制，并进行调整以适应相应的13C或15N掺入。

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Ex vivo and in vivo stable isotope labelling of central carbon metabolism and related pathways with analysis by LC-MS/MS.

Targeted tandem mass spectrometry (LC-MS/MS) has been extremely useful for profiling small molecules extracted from biological sources, such as cells, bodily fluids and tissues. Here, we present a protocol for analysing incorporation of the non-radioactive stable isotopes carbon-13 (13C) and nitrogen-15 (15N) into polar metabolites in central carbon metabolism and related pathways. Our platform utilizes selected reaction monitoring (SRM) with polarity switching and amide hydrophilic interaction liquid chromatography (HILIC) to capture transitions for carbon and nitrogen incorporation into selected metabolites using a hybrid triple quadrupole (QQQ) mass spectrometer. This protocol represents an extension of a previously published protocol for targeted metabolomics of unlabeled species and has been used extensively in tracing the metabolism of nutrients such as 13C-labeled glucose, 13C-glutamine and 15N-glutamine in a variety of biological settings (e.g., cell culture experiments and in vivo mouse labelling via i.p. injection). SRM signals are integrated to produce an array of peak areas for each labelling form that serve as the output for further analysis. The processed data are then used to obtain the degree and distribution of labelling of the targeted molecules (termed fluxomics). Each method can be customized on the basis of known unlabeled Q1/Q3 SRM transitions and adjusted to account for the corresponding 13C or 15N incorporation. The entire procedure takes ~6-7 h for a single sample from experimental labelling and metabolite extraction to peak integration.

更新日期：2019-01-26

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