RESUMO
For the characterization of the metabolic heterogeneity of cell populations, high-throughput single-cell analysis platforms are needed. In this study, we utilized mass spectrometry (MS) enhanced with ion mobility separation (IMS) and coupled with an automated sampling platform, fiber-based laser ablation electrospray ionization (f-LAESI), for in situ high-throughput single-cell metabolomics in soybean (Glycine max) root nodules. By fully automating the in situ sampling platform, an overall sampling rate of 804 cells/h was achieved for high numbers (>500) of tissue-embedded plant cells. This is an improvement by a factor of 13 compared to the previous f-LAESI-MS configuration. By introducing IMS, the molecular coverage improved, and structural isomers were separated on a millisecond time scale. The enhanced f-LAESI-IMS-MS platform produced 259 sample-related peaks/cell, almost twice as much as the 131 sample-related peaks/cell produced by f-LAESI-MS without IMS. Using the upgraded system, two types of metabolic heterogeneity characterization methods became possible. For unimodal metabolite abundance distributions, the metabolic noise reported on the metabolite level variations within the cell population. For bimodal distributions, the presence of metabolically distinct subpopulations was established. Discovering these latent cellular phenotypes could be linked to the presence of different cell states, e.g., proliferating bacteria in partially occupied plant cells and quiescent bacteroids in fully occupied cells in biological nitrogen fixation, or spatial heterogeneity due to altered local environments.
