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Direct metabolomics for plant cells by live single-cell mass spectrometry

Nature Protocols volume 10, pages 1445–1456 (2015)Cite this article

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Abstract

Live single-cell mass spectrometry (live MS) provides a mass spectrum that shows thousands of metabolite peaks from a single live plant cell within minutes. By using an optical microscope, a cell is chosen for analysis and a metal-coated nanospray microcapillary tip is used to remove the cell's contents. After adding a microliter of ionization solvent to the opposite end of the tip, the trapped contents are directly fed into the mass spectrometer by applying a high voltage between the tip and the inlet port of the spectrometer to induce nanospray ionization. Proteins are not detected because of insufficient sensitivity. Metabolite peaks are identified by exact mass or tandem mass spectrometry (MS/MS) analysis, and isomers can be separated by combining live MS with ion-mobility separation. By using this approach, spectra can be acquired in 10 min. In combination with metabolic maps and/or molecular databases, the data can be annotated into metabolic pathways; the data analysis takes 30 min to 4 h, depending on the MS/MS data availability from databases. This method enables the analysis of a number of metabolites from a single cell with rapid sampling at sub-attomolar-level sensitivity.

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Figure 1: Schematic principle of live plant single-cell MS.
Figure 2: Experimental setup.
Figure 3: Isomer separation of catechins from a single plant leaf cell by ion mobility-tripleQ/MS.
Figure 4
Figure 5: Results of live single plant cell MS.
Figure 6: Principal component analyses of live single plant cell mass spectra.
Figure 7: Typical spectra in live single plant cell MS.

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Acknowledgements

This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Grant for Development of Systems and Technology for Advanced Measurement and Analysis by the Japan Science and Technology Agency (JST). We thank A. Makarov (Thermo Scientific) for his kind support in instrumentation using Orbitrap/MS, K. Karasawa (AB Sciex) for support in data analysis, P. Karagiannis (QBiC, RIKEN) for helping with the English in this manuscript and all students (Hiroshima University) and staff (QBiC, RIKEN) who have engaged in this live MS project.

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Authors and Affiliations

  1. Laboratory for Molecular Medicine and Devices, Graduated School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan

    Takashi Fujii, Shuichi Matsuda, Mónica Lorenzo Tejedor, Hajime Mizuno, Naohiro Tsuyama & Tsutomu Masujima

  2. Laboratory for Single Cell Mass Spectrometry, Quantitative Biology Center, RIKEN, Suita, Osaka, Japan

    Takashi Fujii, Shuichi Matsuda, Tsuyoshi Esaki, Iwao Sakane, Hajime Mizuno, Naohiro Tsuyama & Tsutomu Masujima

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Contributions

T.F., S.M., M.L.T., T.E., I.S., H.M. and N.T. carried out experiments and discussed and wrote partial drafts of the manuscript; T.M. designed and directed the study together with necessary instrumentation developments, and rearranged and wrote the final manuscript.

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Correspondence to Tsutomu Masujima.

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41596_2015_BFnprot2015084_MOESM353_ESM.mpg

The trapping of a single plant cell (vacuole of a radish sprout cell) by sucking with a nanospray needle under a stereomicroscope. (MPG 1498 kb)

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Fujii, T., Matsuda, S., Tejedor, M. et al. Direct metabolomics for plant cells by live single-cell mass spectrometry. Nat Protoc 10, 1445–1456 (2015). https://doi.org/10.1038/nprot.2015.084

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