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

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.

Figure 1

Experimental setup of the single-cell CE-ESI-MS system. (a) Front view of the CE platform highlighting (1) the enclosure equipped with a safety-interlock enabled door, (2) platform for sample loading, which can be rapidly elevated by 15 cm (see arrow), (3) holder allowing manual positioning of the separation capillary in three degrees of freedom, (4) separation capillary (solid line in red), (5) resistor connected in series to a stable HVPS (Figure S2) and the CE platform, and (6) digital multimeter connected in parallel measuring voltage drop on the resistor. Scale = 10 cm. (b) Magnified view of the sample-loading platform consisting of (7) the sample-loading vial and (8) electrolyte-containing vial with the separation capillary positioned 2 mm below the electrolyte meniscus. Scale = 1 cm. (c) Distant view of the CE-ESI-MS ion source consisting of (9) the CE-ESI interface mounted on (10) the three-axis translation stage of a PicoView nanospray source, (11) CCD camera equipped with light-collimating and focusing lenses to record ES performance, and (12) a mass spectrometer equipped with a nanospray sampling plate. (d) Close-up view of the CE-ESI-MS ion source highlighting a T-union that houses fused silica capillaries for (4) CE separation and (13) ES sheath solution delivery as well as (14) a metal emitter grounded (earth) through a (15) thin copper wire. Scale = 1 cm. (e) Magnified view of (16) the stable Taylor-cone formed upon operating the ES in the cone-jet spraying mode (see reflected image) in front of (17) the orifice of the mass spectrometer sampling plate. Scale = 500 µm.

Figure 2

Targeted CE-ESI-MS analysis of single neurons, 25–500 µm in diameter. (a) Optical images show (left panel) left pleural 1 (LPl1), R2, and R15 abdominal, and metacerebral (MCC) neurons, 150–350 µm in cell body diameter, located in the corresponding ganglia from the A. californica CNS, with other neurons and nerves located adjacently, and (right panel) a DRG neuron, 25 µm in cell body diameter, from the rat peripheral nervous system. Scales = 1 mm and 20 µm, respectively. (b) Multifaceted data analysis identifies a molecular feature for the ion with a nominal mass of m/z 182 ± 0.5 in the MCC neuron extract and determines the corresponding accurate mass of the compound (right insets, bgr = background spectrum). Comparison of the data against a mass spectrometric metabolite database yields tyrosine as a putative identification (based on accurate mass). Confirmation via tandem mass spectrometric and migration time measurements on the related standard (lower spectra) corroborates the finding. Migration times are aligned using internal standards and a quadratic equation. (c) In a targeted analysis, selected metabolites are profiled in single LPl1, MCC, and R15 neurons, and a 25-µm-diameter DRG neuron.

Figure 3

Chemical profiling, quantitation, and comparison of metabolites among individual cells. (a) (Top panel) The unsupervised PCA score plot indicates metabolic differences between MCC, R15, B1 or B2, and R2 or LPl1 single neurons and uncovers similarities between B1 and B2 as well as R2 and LPl1 cells. Underlined numbers correspond to metabolites identified per reference and include histamine (1), acetylcholine (9), serotonin (13), glycine betaine (32), and proline betaine (33). (Bottom panel) The corresponding loading plot reveals metabolites with different abundance patterns among the extracts. (b) Student’s t-test identifies statistically significant differences in the acetylcholine, serotonin, and tyrosine levels measured in LPl1, MCC, and R15 single neurons; two asterisks (**) mark p < 0.005. (c) External concentration calibration with serine standard helps to quantify this endogenous metabolite in selected LPl1 and R15 neurons. Differences in serine concentration are apparent among the cell types as well as individual neurons of the same type. (Adapted with permission from reference. Copyright 2011 American Chemical Society.)