Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry

Abstract

The use of single-cell assays has emerged as a cutting-edge technique during the past decade. Although single-cell mass spectrometry (MS) has recently achieved remarkable results, deep biological insights have not yet been obtained, probably because of various technical issues, including the unavoidable use of matrices, the inability to maintain cell viability, low throughput because of sample pretreatment, and the lack of recordings of cell physiological activities from the same cell. In this study, we describe a patch clamp/MS-based platform that enables the sensitive, rapid, and in situ chemical profiling of single living neurons. This approach integrates modified patch clamp technique and modified MS measurements to directly collect and detect nanoliter-scale samples from the cytoplasm of single neurons in mice brain slices. Abundant possible cytoplasmic constituents were detected in a single neuron at a relatively fast rate, and over 50 metabolites were identified in this study. The advantages of direct, rapid, and in situ sampling and analysis enabled us to measure the biological activities of the cytoplasmic constituents in a single neuron, including comparing neuron types by cytoplasmic chemical constituents; observing changes in constituent concentrations as the physiological conditions, such as age, vary; and identifying the metabolic pathways of small molecules.single neuron | mass spectrometry | metabolism | patch clamp

Confined Nanopipette Sensing: From Single Molecules, Single Nanoparticles, to Single Cells

“…[81] Moreover,b yi ntegrating nano-ESI pipettes in the patch clamp platform, single living neurons have been profiled rapidly using only nanoliter-scale samples. [82] As aconsequence of the sample collection features,nanopipettes were further used in matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS). Them etabolites in cytoplasm have been characterized as shown in Figure 5 (signals labeled with ar ed star).…”

Single‐Cell Mass Spectrometry Approaches to Explore Cellular Heterogeneity

“…[97] Thes ingle probe is ap ulled double-barrel capillary, where one of the barrels is continuously supplied with electrospray solution and the other barrel is connected to ananospray emitter. [99] Theu nique combination of these two technologies enabled the identification of more than 50 metabolites in the cytoplasm of mouse brain neurons,a nd captured variations between different neuron types and mice of differing ages.P airing electrophysiology with MS analysis for asingle neuron presents great potential for molecular neuroscience. This technique has been applied to the detection of adenylates,l ipids,a nd drug molecules in individual HeLa cells treated with anticancer pharmaceuticals.…”

“…[97][98] To simultaneously capture the metabolic and electrophysiological state of aneuron, the nano-ESI-MS method was combined with the patch-clamp technique. [99] Theu nique combination of these two technologies enabled the identification of more than 50 metabolites in the cytoplasm of mouse brain neurons,a nd captured variations between different neuron types and mice of differing ages.P airing electrophysiology with MS analysis for asingle neuron presents great potential for molecular neuroscience.…”

Erforschung der fundamentalen Strukturen des Lebens: Nicht zielgerichtete chemische Analyse von Einzelzellen und subzellulären Strukturen

“…Vertes und Mitarbeiter [101] kombinierten im Jahr 2018 Fluoreszenzmikroskopie mit kapillarer Mikroprobenahme, um eine ESI-Analyse von Zellen in verschiedenen mitotischen Stadien durchführen. Huang et al [102] [103] haben über 700 Analyten innerhalb einer einzelnen Zelle mit Lebendzell-MS entdeckt und diese Te chnik zur Analyse von Metaboliten in Pflanzen verwendet. [104] In ähnlicher Weise demonstrierten Huang et al [105] die Analyse von Proteinen aus lebenden Zellen, ohne die Zelle zu zerstçren.…”