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Discovery and neurochemical screening of peptides in brain extracellular fluid by chemical analysis of in vivo microdialysis samples.

Endogenous peptides from brain extracellular fluid of live rats were analyzed using capillary liquid chromatography (LC)-tandem mass spectrometry (MS2). A 4-mm-long microdialysis probe perfused at 0.6 microL/min implanted into the striatum of anesthetized male rats was used to collect 3.6 microL dialysate fractions that were injected on-line into the capillary LC-MS2 system for analysis. A total of 3349 MS2 spectra were collected from 13 different animals under basal conditions and during localized depolarization evoked by infusion of a high-K+ solution through the microdialysis probe. Subtractive analysis revealed a total of 859 MS2 spectra that were observed only during depolarization. From these spectra, 29 peptide sequences (25 were peptides not previously observed) from 6 different protein precursors were identified using database searching software. Proteins identified include precursors to neuropeptides, synaptic proteins, blood proteins, and transporters. The identified peptides represent candidates for neurotransmitters, neuromodulators, and markers of synaptic activity or brain tissue damage. A screen for neuroactivity of novel proenkephalin fragments that were found was performed by infusing the peptides into the brain while monitoring amino acid neurotransmitters by microdialysis sampling combined with capillary electrophoresis. Three of the six tested peptides evoked significant increases in various neuroactive amino acids. These results demonstrate that this combination of methods can identify novel neurotransmitter candidates and screen for potential neuroactivity.