Patch-Clamp Single-Cell Proteomics in Acute Brain Slices: A Framework for Recording, Retrieval, and Interpretation

Area of Science:

• Neuroscience
• Molecular Biology
• Proteomics

Background:

• Single-cell proteomics (SCP) offers insights into neuronal molecular composition but is limited in acute brain slices.
• Patch-clamp electrophysiology complements SCP by assessing neuronal excitability and function.
• Combining these techniques presents challenges in physiological characterization and physical collection of neurons.

Purpose of the Study:

• To develop a framework for interpreting patch-SCP outcomes considering retrieval quality.
• To benchmark the retrieval of pyramidal neurons from rat medial prefrontal cortex using a shotgun SCP approach.
• To establish a practical method for linking neuronal physiology with proteomics in semi-intact circuits.

Main Methods:

• Utilized a shotgun strategy, collecting all patched neurons regardless of electrophysiological outcome.
• Systematically benchmarked neuron retrieval from rat medial prefrontal cortex slices.
• Correlated electrophysiological parameters (capacitance, spiking) with proteomic yield and synaptic content.

Main Results:

• Neuronal capacitance during gigaseal-preserved retrieval correlated with protein identification, linking soma size to proteome yield.
• Preservation of neuronal spiking during relocation correlated with synaptic enrichment and recovery of transmembrane proteins.
• Torn or aspirated neurons yielded smaller proteomes with poor synaptic representation, while lost gigaseal neurons showed variable but substantial molecular data.

Conclusions:

• Shotgun patch-SCP provides a proof-of-concept and practical framework for integrating neuronal physiology and proteomics.
• Retrieval quality, assessed by material amount and synaptic content, is crucial for interpreting patch-SCP results.
• This approach enables linking detailed physiological measurements with molecular profiles in semi-intact neuronal circuits.