Samarah Laith Z, Vertes Akos, Anderton Christopher R
Department of Chemistry, George Washington University, Washington, DC, USA.
Environmental Molecular Sciences Laboratory and Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA.
Methods Mol Biol. 2022;2437:61-75. doi: 10.1007/978-1-0716-2030-4_4.
Metabolomic measurements can provide functional readouts of cellular states and phenotypes. Here, we present a protocol for single-cell metabolomics that permits direct untargeted detection of a broad number of metabolites under ambient conditions, without the need for sample processing, and with high confidence in the discovery and identification of the molecular formulas for detected metabolites. This protocol describes combining fiber-based laser ablation electrospray ionization (f-LAESI) with a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer (21T-FTICR-MS) to obtain high confidence molecular formula information about detected metabolites. The f-LAESI source utilizes mid-infrared laser ablation through a sharp optical fiber tip, affording direct ambient analysis of cells without the need for sample processing. Using the 21T-FTICR-MS as a mass analyzer enabled measurement of the isotopic fine structure (IFS) for numerous metabolites simultaneously from single cells, and the IFSs were in turn computationally processed to rapidly determine the corresponding elemental compositions. This metabolomics technique complements other single cell omics measurement methods, helping to resolve complex molecular interactions that take place within cells unattainable from single cell transcriptomic and proteomics methods.
代谢组学测量能够提供细胞状态和表型的功能读数。在此，我们展示了一种单细胞代谢组学方案，该方案允许在环境条件下直接对大量代谢物进行非靶向检测，无需样品处理，并且在发现和鉴定所检测代谢物的分子式方面具有高度可信度。本方案描述了将基于光纤的激光烧蚀电喷雾电离（f-LAESI）与一台21特斯拉傅里叶变换离子回旋共振质谱仪（21T-FTICR-MS）相结合，以获取有关所检测代谢物的高可信度分子式信息。f-LAESI源通过尖锐的光纤尖端利用中红外激光烧蚀，无需样品处理即可对细胞进行直接环境分析。使用21T-FTICR-MS作为质量分析器能够同时测量来自单细胞的多种代谢物的同位素精细结构（IFS），然后对这些IFS进行计算处理以快速确定相应的元素组成。这种代谢组学技术补充了其他单细胞组学测量方法，有助于解析单细胞转录组学和蛋白质组学方法无法实现的细胞内发生的复杂分子相互作用。
