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

Understanding of the relationship between cellular function and molecular composition holds a key to next-generation therapeutics but requires measurement of all types of molecules in cells. Developments in sequencing enabled semiroutine 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 similar to 0.25% of the total cellular proteome, was analyzed on a custom-built capillary electrophoresis (CE) electrospray ionization platform using orbitrap HRMS for detection. A series of experiments were conducted to systematically enhance detection sensitivity through refinements in sample processing and detection, allowing us to quantify similar to 275 different proteins from somal aspirate-equivalent protein digests from cultured neurons. From single neurons, patch-clamp proteomics of the soma quantified 91, 80, and 95 different proteins from three 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 observed proteins. The integration of patch-clamp electrophysiology with subcellular CE-HRMS proteomics expands the analytical toolbox of neuroscience.