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

Single-cell proteomics (SCP) is a powerful method for interrogating the molecular composition of neurons, yet its application to acute brain slices has remained limited. Patch-clamp electrophysiology provides direct information on neuronal excitability, synaptic inputs, and ion channel function, making it a natural partner for SCP. However, combining these techniques introduces unique challenges: neurons must be both physiologically characterized and physically collected, and variability during retrieval from the brain slice can affect how faithfully proteomic measurements reflect <i>in situ</i> physiology. Here, we introduce a framework for interpreting patch-SCP outcomes that considers retrieval quality in terms of both the amount of material collected and the synaptic contents being recovered. Using a shotgun strategy in which all patched neurons were collected regardless of electrophysiological outcome, we systematically benchmarked the retrieval of pyramidal neurons in the rat medial prefrontal cortex. Capacitance measured during gigaseal-preserved retrieval correlated with protein identifications, providing a proxy for linking soma size to proteome yield. Preservation of neuronal spiking during relocation was associated with broader synaptic enrichment and recovery of transmembrane proteins. By comparison, torn or aspirated neurons produced small proteomes with poor synaptic representation and neurons with lost gigaseals or no recordings displayed variable outcomes that could still yield substantial molecular information. Together, these results establish shotgun patch-SCP as both proof-of-concept and a practical framework for linking neuronal physiology with proteomics in semi-intact circuits.