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Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry

Nature Protocols volume 8, pages 783–799 (2013)Cite this article

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Abstract

Single-cell mass spectrometry (MS) empowers metabolomic investigations by decreasing analytical dimensions to the size of individual cells and subcellular structures. We describe a protocol for investigating and quantifying metabolites in individual isolated neurons using single-cell capillary electrophoresis (CE) coupled to electrospray ionization (ESI) time-of-flight (TOF) MS. The protocol requires ∼2 h for sample preparation, neuron isolation and metabolite extraction, and 1 h for metabolic measurement. We used the approach to detect more than 300 distinct compounds in the mass range of typical metabolites in various individual neurons (25–500 μm in diameter) isolated from the sea slug (Aplysia californica) central and rat (Rattus norvegicus) peripheral nervous systems. We found that a subset of identified compounds was sufficient to reveal metabolic differences among freshly isolated neurons of different types and changes in the metabolite profiles of cultured neurons. The protocol can be applied to the characterization of the metabolome in a variety of smaller cells and/or subcellular domains.

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Figure 1: Experimental setup of the single-cell CE-ESI-MS system.
Figure 2: Targeted CE-ESI-MS analysis of single neurons, 25–500 μm in diameter.
Figure 3: Chemical profiling, quantification and comparison of metabolites among individual cells.

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Acknowledgements

T. Lapainis contributed to the development of the CE-ESI-MS platform, X. Wang facilitated the single-neuron preparation work and A.M. Knolhoff helped with the interpretation of CE-ESI-MS data in the original CE-MS studies. This work was supported by the National Science Foundation under award no. CHE 11-11705, the National Institute of Neurological Disorders and Stroke under award no. NS031609, the National Institute of Dental and Craniofacial Research and the Office of the Director, US National Institutes of Health under award no. DE018866 and the National Institute on Drug Abuse under award no. P30 DA018310. The mention of commercial products, their sources or their use in connection with material reported herein is not to be construed as either actual or implied endorsement of such products by the Department of Health and Human Services. The authors thank S. Baker for assistance in the preparation of this publication and the machine shop of the School of Chemical Sciences, University of Illinois at Urbana-Champaign, for providing various components for the construction of the CE-ESI system. They also thank M. Kim for help in photography.

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  1. Peter Nemes

    Present address: Present address: US Food and Drug Administration, Silver Spring, Maryland, USA.,

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  1. Department of Chemistry and the Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Peter Nemes, Stanislav S Rubakhin, Jordan T Aerts & Jonathan V Sweedler

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Contributions

The contributions of the authors to the original studies that were used to generate the protocol are indicated in the original cited articles. For the protocols described here, P.N. generated many of the protocol steps related to CE-MS operation and wrote the data analysis script and virtual instrument; J.T.A. modified the protocols and validated them using the DRG neurons; S.S.R. performed the cell isolations and optimized the protocols for cell handling; and J.V.S. modified and clarified the protocol. All authors participated in the writing of the protocol.

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Correspondence to Jonathan V Sweedler.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data

Supplementary sequence archive (ZIP 120 kb)

Supplementary Methods

Stable ion generation, reproducible separation, and semiautomated data analysis (PDF 901 kb)

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Nemes, P., Rubakhin, S., Aerts, J. et al. Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry. Nat Protoc 8, 783–799 (2013). https://doi.org/10.1038/nprot.2013.035

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