State Key Laboratory of Organometallic Chemistry and National Center for Organic Mass Spectrometry in Shanghai, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032, China.
Anal Chem. 2020 Jun 16;92(12):8378-8385. doi: 10.1021/acs.analchem.0c00954. Epub 2020 May 29.
Currently in single-cell mass spectrometry, the analysis of low-abundance cell metabolites such as fatty alcohols and sterols remains a challenge. In most research studies, single-cell samples are analyzed directly after sampling. However, this workflow may exclude many effective sample pretreatment methods such as derivatization for the improvement of detection sensitivity for specific cell metabolites in a single-cell sample. Metabolites in low abundance in a cell may not be detected. Herein on-probe derivatization coupled with noncontact nanocarbon fiber ionization is proposed for sensitive fatty alcohol and sterol metabolite analysis at the single-cell level. Fatty alcohol and sterol metabolites were rapidly quaternized by the single-cell on-probe derivatization method. The reaction products were directly ionized with no postreaction processing. Furthermore, a new ionization source for noncontact nanocarbon fiber ionization was developed to show good compatibility with dichloromethane, a low-polarity solvent used in on-probe derivatization. The quaternized fatty alcohols and sterols exhibited evidently enhanced ionization efficiency in mass spectra. In applications of the developed method, seven kinds of even-numbered-carbon fatty alcohols (C-C) and five kinds of sterols were detected in single L-02 and HepG2 cells. Then the L-02 and HepG2 cells were readily discriminated through principal component analysis. Additionally, a rough quantitative analysis of the detected fatty alcohols and sterols in single cells was performed. The mass intensities of fatty alcohols show a significant difference between L-02 and HepG2 cells while those of sterols remain stable.
目前，单细胞质谱分析中，对低丰度细胞代谢物（如脂肪醇和固醇）的分析仍然是一个挑战。在大多数研究中，单细胞样品都是在取样后直接进行分析。然而，这种工作流程可能会排除许多有效的样品预处理方法，如衍生化，以提高单细胞样品中特定细胞代谢物的检测灵敏度。细胞中低丰度的代谢物可能无法检测到。在此，提出了一种在单细胞水平上对低丰度脂肪醇和固醇代谢物进行灵敏分析的探针衍生化结合非接触纳米碳纤维离子化方法。通过单细胞探针衍生化方法，脂肪醇和固醇代谢物迅速季铵化。反应产物无需后处理即可直接离子化。此外，还开发了一种新的非接触纳米碳纤维离子源，与探针衍生化中使用的低极性溶剂二氯甲烷具有良好的兼容性。季铵化的脂肪醇和固醇在质谱中表现出明显增强的离子化效率。在开发方法的应用中，在单个 L-02 和 HepG2 细胞中检测到七种偶数碳原子脂肪醇（C-C）和五种固醇。然后通过主成分分析很容易区分 L-02 和 HepG2 细胞。此外，还对单细胞中检测到的脂肪醇和固醇进行了粗略的定量分析。脂肪醇的质量强度在 L-02 和 HepG2 细胞之间存在显著差异，而固醇的质量强度保持稳定。
