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

2021 ◽  
Author(s):  
Sam B. Choi ◽  
Abigail M. Polter ◽  
Peter Nemes

ABSTRACTUnderstanding 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.

2017 ◽  
Vol 951 ◽  
pp. 140-150 ◽  
Author(s):  
Syntia Fayad ◽  
Reine Nehmé ◽  
Monika Langmajerová ◽  
Benjamin Ayela ◽  
Cyril Colas ◽  
...  

2018 ◽  
Vol 91 (2) ◽  
pp. 1295-1301 ◽  
Author(s):  
Kazunori Sasaki ◽  
Hitoshi Sagawa ◽  
Makoto Suzuki ◽  
Hiroyuki Yamamoto ◽  
Masaru Tomita ◽  
...  

2018 ◽  
Vol 547 ◽  
pp. 14-18 ◽  
Author(s):  
Sébastien Acket ◽  
Anthony Degournay ◽  
Mathilde Gosset ◽  
Franck Merlier ◽  
Manual Adrian Troncoso-Ponce ◽  
...  

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