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. For instance,
after patch-clamping a neuron, its soma must be physically retrieved,
and variability during extraction from the brain slice may influence
how well proteomic measurements reflect in situ physiology.
Here, we introduce a framework for contextualizing patch-SCP outcomes,
with an emphasis on retrieval quality (material yield and soma-enriched
synaptic content). We used an indiscriminate shotgun strategy in which
all patched neurons were collected regardless of electrophysiological
outcome to assess soma retrieval in an exploratory data set of rat
medial prefrontal cortex pyramidal neurons. Capacitance during gigaseal-preserved
retrieval correlated with protein identifications, suggesting that
proteome yield could be linked to soma size. Preservation of neuronal
spiking during relocation tended to be 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 little to no characterization displayed
more variable outcomes. These results demonstrate that patch-SCP can
be used to assess soma retrieval and they provide a framework for
interpreting how electrophysiological context and soma retrieval quality
shape single-neuron proteomic measurements in semi-intact circuits.