Neuropeptides in the Extracellular Space of the Mouse Cortex Measured In Vivo by Nanodialysis Probe Coupled with LC‐MS

Neuropeptides are key neuromodulators in the central nervous system that shape sensory processing, yet their extracellular dynamics in the somatosensory cortex (S1) remain poorly understood. This study applies an innovative membrane‐free silicon nanodialysis (ND) probe coupled with liquid chromatography‐mass spectrometry (LC‐MS) to analyze the extracellular neuropeptidome in the mouse S1 with spatial resolution down to 100 µm. Localized in vivo sampling identified extracellular peptides from secretogranin‐1, ProSAAS, pro‐opiomelanocortin (POMC), and others. Minimal tissue damage, enabled by probe dimensions of 75 × 15 µm 2 , resulted in an absence of structural peptides in the dialysate indicating low intracellular contamination. Many detected secretory peptides correlated with strong local mRNA expression; however, the detection of POMC‐derived peptides, despite negligible local expression, suggests long‐distance peptide transport or extracellular processing. To expand peptide identification, a discovery‐to‐targeted peptidomic approach was developed, revealing 46 peptides from 24 proteins in dialysate samples, including 10 proteins with low local expression. Complementary S1 tissue analysis confirmed POMC peptides and showed that 17% of 304 prohormone‐derived peptides had low local expression. These results uncover a complex extracellular peptide landscape shaped by both local and long‐distance signaling. By overcoming the limitations of traditional microdialysis, this approach advances the understanding of neuropeptide signaling in cortical function.