Exploring peptides in the brain by in vivo microdialysis-capillary liquid chromatography-tandem mass spectrometry

Neuropeptides are hormones, neurotransmitters, and neuromodulators important in learning, memory, appetite regulation, sensory perception and disease. Many recent studies have shown that post-translational proteolytic processing produces neuropeptides that were not predicted from known enzymatic cleavage sites. Thus, the mechanism for regulating the bioactivity and stoichiometry of peptide products from protein precursors remains a mystery. In this work is described the development and application of novel bioanalytical methods and tools for monitoring and discovering endogenous neuropeptides in the mammalian brain.; Time-segmented and data-dependent tandem mass spectra were collected from male Sprague-Dawley rats during basal and K+-induced depolarizing conditions. A 4 mm microdialysis probe sampled the globus pallidus or striatum region of the rat brain at 0.6 μL/min for on-line, in vivo microdialysis-capillary liquid chromatography-tandem mass spectrometry. Multiple peptides at low attomole levels (low picomolar concentrations) were quantified and sequenced in microdialysate samples by using 25 μm i.d. columns with integrated 3 μm i.d. electrospray emitters, optimized gradients and a quadrupole ion trap mass spectrometer.; This is the first report of on-line in vivo microdialysis of endogenous neuropeptides in combination with (1) separation and simultaneous monitoring, (2) data-dependent measurement, (3) mass spectral library construction and (4) novel peptide discovery. The discovery of endogenous neuropeptides provides new insights into inter-neuron chemical signaling. This method should be useful for monitoring known peptides, studying processing of endogenous peptides from protein precursors and characterizing novel peptides that are potentially bioactive. Moreover, exploring the extracellular release of endogenous neuropeptides suggests novel biomarkers for biomedical applications.