In situ capillary microsampling with capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry (MS) enabled the characterization of cationic metabolites in single cells in complex tissues and organisms. For deeper coverage of the metabolome and metabolic networks, analytical approaches are needed that provide complementary detection for anionic metabolites, ideally using the same instrumentation. Described here is one such approach that enables sequential cationic and anionic (dual) analysis of metabolites in the same identified cell in a live vertebrate embryo. A calibrated volume was microaspirated from the animal-ventral cell in a live 8-cell embryo of Xenopus laevis , and cationic and anionic metabolites were one-pot microextracted from the aspirate, followed by CE-ESI-MS analysis of the same extract. A laboratory-built CE-ESI interface was reconfigured to enable dual cationic–anionic analysis with ∼5–10 nM (50–100 amol) lower limit of detection and a capability for quantification. To provide robust separation and efficient ion generation, the CE-ESI interface was enclosed in a nitrogen gas filled chamber, and the operational parameters were optimized for the cone-jet spraying regime in both the positive and negative ion mode. A total of ∼250 cationic and ∼200 anionic molecular features were detected from the cell between m / z 50–550, including 60 and 24 identified metabolites, respectively. With only 11 metabolites identified mutually, the duplexed approach yielded complementary information on metabolites produced in the cell, which in turn deepened network coverage for several metabolic pathways. With scalability to smaller cells and adaptability to other types of tissues and organisms, dual cationic–anionic detection with in situ microprobe CE-ESI-MS opens a door to better understand cell metabolism.
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Single-cell mass-spectrometry of metabolites and proteins for systems and functional biology, in: Single Cell ‘Omics of Neuronal Cells
Molecular composition is intricately intertwined with cellular function, and elucidation of this relationship is essential for understanding life processes and developing next-generational therapeutics. Technological innovations in capillary electrophoresis (CE) and liquid chromatography (LC)-mass spectrometry (MS) provide previously unavailable insights into cellular biochemistry by allowing for the unbiased detection and quantification of molecules with high specificity. This chapter presents our validated protocols integrating ultrasensitive MS with classical tools of cell, developmental, and neurobiology to assess the biological function of important biomolecules. We use CE- and LC-MS to measure hundreds of metabolites and thousands of proteins in single cells or limited populations of tissues in chordate embryos and mammalian neurons, revealing molecular heterogeneity between identified cells. By pairing microinjection and optical microscopy, we enable cell lineage tracing and testing of the roles that dysregulated molecules play in the formation and maintenance of cell heterogeneity and tissue specification in frog embryos (Xenopus laevis). Electrophysiology extends our workflows to characterizing neuronal activity in sections of mammalian brain tissues. The information obtained from these studies mutually strengthen chemistry and biology and highlight the importance of interdisciplinary research to advance basic knowledge and translational applications forward.
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- Award ID(s):
- 1832968
- NSF-PAR ID:
- 10422238
- Editor(s):
- Sweedler, J. V.; Eberwine, J. H.; Fraser, S. E.
- Date Published:
- Journal Name:
- Neuromethods
- Volume:
- 184
- ISSN:
- 1940-6045
- Page Range / eLocation ID:
- 87–114
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract We report the development of in vivo subcellular high‐resolution mass spectrometry (HRMS) for proteo‐metabolomic molecular systems biology in complex tissues. With light microscopy, we identified the left‐dorsal and left‐ventral animal cells in cleavage‐stage non‐sentient Xenopus laevis embryos. Using precision‐translated fabricated microcapillaries, the subcellular content of each cell was double‐probed, each time swiftly (<5 s/event) aspirating <5 % of cell volume (≈10 nL). The proteins and metabolites were analyzed by home‐built ultrasensitive capillary electrophoresis electrospray ionization employing orbitrap or time‐of‐flight HRMS. Label‐free detection of ≈150 metabolites (57 identified) and 738 proteins found proteo‐metabolomic networks with differential quantitative activities between the cell types. With spatially and temporally scalable sampling, the technology preserved the integrity of the analyzed cells, the neighboring cells, and the embryo. 95 % of the analyzed embryos developed into sentient tadpoles that were indistinguishable from their wild‐type siblings based on anatomy and visual function in a background color preference assay.
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Abstract We report the development of in vivo subcellular high‐resolution mass spectrometry (HRMS) for proteo‐metabolomic molecular systems biology in complex tissues. With light microscopy, we identified the left‐dorsal and left‐ventral animal cells in cleavage‐stage non‐sentient Xenopus laevis embryos. Using precision‐translated fabricated microcapillaries, the subcellular content of each cell was double‐probed, each time swiftly (<5 s/event) aspirating <5 % of cell volume (≈10 nL). The proteins and metabolites were analyzed by home‐built ultrasensitive capillary electrophoresis electrospray ionization employing orbitrap or time‐of‐flight HRMS. Label‐free detection of ≈150 metabolites (57 identified) and 738 proteins found proteo‐metabolomic networks with differential quantitative activities between the cell types. With spatially and temporally scalable sampling, the technology preserved the integrity of the analyzed cells, the neighboring cells, and the embryo. 95 % of the analyzed embryos developed into sentient tadpoles that were indistinguishable from their wild‐type siblings based on anatomy and visual function in a background color preference assay.
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null (Ed.)Background Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive. Methods To identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling. Results With the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107a pos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice. Conclusions Broadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.more » « less
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