Patch-Clamp Proteomics of Single Neuronal Somas in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry

Abstract

Understanding of the relationship between cellular function and molecular composition holds a key to next-generational therapeutics but requires measurement of all types of molecules in cells. Developments in sequencing enabled semi-routine measurement of single-cell genomes and transcriptomes, but analytical tools are scarce for detecting diverse proteins in tissue-embedded cells. To bridge this gap for neuroscience research, we report the integration of patch-clamp electrophysiology with subcellular shot-gun proteomics by high-resolution mass spectrometry (HRMS). Recording of electrical activity permitted identification of dopaminergic neurons in the substantia nigra pars compacta. Ca. 20-50% of the neuronal soma content, containing an estimated 100 pg of total protein, was aspirated into the patch pipette filled with ammonium bicarbonate. About ~1 pg of somal protein, or ~0.25% of the total cellular proteome, was analyzed on a custom-built capillary electrophoresis (CE) high-resolution mass spectrometer (HRMS). A series of experiments were conducted to systematically enhance detection sensitivity through refinements in sample processing and detection, allowing us to quantify ~275 different proteins from somal aspirate-equivalent protein digests. From single neurons, patch-clamp proteomics of the soma quantified 91, 80, and 95 different proteins from 3 different dopaminergic neurons, or 157 proteins in total. Quantification revealed detectable proteomic differences between the somal protein samples. Analysis of canonical knowledge predicted rich interaction networks between the proteins. The integration of patch-clamp electrophysiology with subcellular CE-HRMS proteomics expands the analytical toolbox of neuroscience.

Patch-Clamp Proteomics of Single Neurons in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry

“…These instruments preserved cell and embryonic viability, allowing for evaluations of anatomy and whole-organismal behavior . In a series of studies, we also found the technology scalable to smaller cells and other functional measurements, including electrical recording of neuronal activity. − This report captures the development, testing, and validation of this technology following the systematic approach outlined in Figure .…”

“…In this study, we expanded the analytical toolbox of neuroscience by integrating patch-clamp electrophysiology with subcellular proteomics using bottom-up CE-HRMS. Since inception in 2019, − this approach has undergone performance improvements that are now sufficient to support neuroscience research. Recording of neuronal activity aided functional characterization of DA neurons in sections of the substantia nigra pars compacta from the mouse brain.…”

“…Recently, we custom-built CE–HRMS platforms with a capability for ∼250 zmol lower limit of detection for model peptides. , These instruments supported the identification of ∼200–800 proteins in protein digests estimating to ∼5–10 neurons. ,, A data-dependent acquisition ladder improved the duty cycle economy, identifying 428 proteins from single-cell equivalent protein amounts from cultured neurons . We recently explored the technical possibility to combine electrophysiology with single-neuron CE–HRMS proteomics. − …”

“…Here, we present the systematic study that enabled the combination of patch-clamp electrophysiology with subcellular CE-ESI-HRMS to quantify proteins in identified neurons in brain tissues. Inspired by the success of patch-clamp single-cell transcriptomics, , peptidomics, and metabolomics, − in 2019, we reported the use of a patch pipette as a microprobe to bridge electrophysiology with microsampling for proteomics. − Here, we report additional refinements, testing, and validations of the method to enhance performance metrics. The approach was developed using dopaminergic (DA) neurons in sections of the substantia nigra pars compacta (mouse) because these cells were readily identifiable based on size, location, and electrical activity.…”

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“…While these methods rely on traditional LC-MS, other groups have explored alternative techniques to further intensify peptide signal. Choi et al described a custom-built capillary electrophoresis (CE) ionization platform using high-resolution mass spectrometry (HRMS) (Choi et al, 2021) (Figure 1C). This platform combines patch-clamp electrophysiology with subcellular CE-ESI-HRMS to quantify proteins in targeted neurons in brain tissues.…”