Patch Clamp Electrophysiology and Capillary Electrophoresis–Mass Spectrometry Metabolomics for Single Cell Characterization

Abstract

The visual selection of specific cells within an ex vivo brain slice, combined with whole-cell patch clamp recording and capillary electrophoresis (CE)–mass spectrometry (MS)-based metabolomics, yields high chemical information on the selected cells. By providing access to a cell’s intracellular environment, the whole-cell patch clamp technique allows one to record the cell’s physiological activity. A patch clamp pipet is used to withdraw ∼3 pL of cytoplasm for metabolomic analysis using CE–MS. Sampling the cytoplasm, rather than an intact isolated neuron, ensures that the sample arises from the cell of interest and that structures such as presynaptic terminals from surrounding, nontargeted neurons are not sampled. We sampled the rat thalamus, a well-defined system containing gamma-aminobutyric acid (GABA)-ergic and glutamatergic neurons. The approach was validated by recording and sampling from glutamatergic thalamocortical neurons, which receive major synaptic input from GABAergic thalamic reticular nucleus neurons, as well as neurons and astrocytes from the ventral basal nucleus and the dorsal lateral geniculate nucleus. From the analysis of the cytoplasm of glutamatergic cells, approximately 60 metabolites were detected, none of which corresponded to the compound GABA. However, GABA was successfully detected when sampling the cytoplasm of GABAergic neurons, demonstrating the exclusive nature of our cytoplasmic sampling approach. The combination of whole-cell patch clamp with single cell cytoplasm metabolomics provides the ability to link the physiological activity of neurons and astrocytes with their neurochemical state. The observed differences in the metabolome of these neurons underscore the striking cell to cell heterogeneity in the brain.

Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo

“…To this end, micromanipulation raises great benefits to tailor single-cell MS to a broader range of applications. Direct sampling of single cells by microcapillaries, as recently demonstrated for neurons (42) and mammalian cells (25), offers an attractive means to enhance the throughput and the precision of measurements on single cells in an embryo using the technique presented here. By providing complementary information to already available genomic, transcriptomic, and proteomic data, we anticipate that small-molecular measurements by single-cell CE-μESI-MS will facilitate basic and translational research in cell and developmental biology to help develop a holistic understanding of how cells acquire their differentiated state.…”

Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS)

“…To this end, we demonstrated the detection/ quantification of a considerable number of proteins from ϳ20 ng protein digests from single blastomeres, approaching the total protein content of larger mammalian cells. Microscale sample preparation can help collect peptides and proteins with high sensitivity, using, for example, patch-clamp electrophysiological tools (62) and microanalysis probes (17,63). For smaller mammalian cells containing fewer amounts of proteins, addition of carrier proteins may be beneficial to minimize adsorptive losses for low-abundance proteins.…”

Capillary electrophoresis mass spectrometry based metabolomics

“…Soga and other labs have clearly demonstrated that most of these early issues and concerns have been overcome by technological improvements, hence the growing list of publications in this field since 2002. CE-MS protocols have been developed to support metabolic measurements, both relative and quantitative, in a large variety of sample types including urine [4,5,8], bacteria [6], CSF [9], neurons [10] and brain [12].…”