Nano liquid chromatography tandem mass spectrometry for the monitoring of hippocampal neuropeptides, possibly involved in limbic epilepsy.

Neuropeptides are an important and diverse class of neuronal signalling molecules. They seem to play an important role when the central nervous system is challenged, as during seizures [1,2]. In order to further characterise the neuropeptides involved in the physiopathology of epilepsy, it is essential to monitor their concentration in the brain. Quantification of neuropeptides in dialysates is challenging due to their low extracellular concentrations (low pM range), their low microdialysis efficiencies and the tendency of peptides to stick to glass and polymeric materials [3]. The quantification of neuropeptides in dialysates therefore necessitates the use of a very sensitive nano LC-MS/MS method. In this study the feasibility of quantifying eight neuropeptides, possibly involved in epilepsy, is investigated. The following neuropeptides were selected for screening, based on known anticonvulsant action and/or their presence in the hippocampus: bradykinin, dynorphin A(1-13), galanin, ghrelin, neuromedin B, neuromedin N, neuropeptide Y, and neurotensin.
First, the MS/MS parameters of the different neuropeptides were optimised by infusing them directly into the nanosource. The following compound-specific MS/MS parameters were obtained: precursor and product ion, cone voltage and collision energy.
Second, all peptides were injected onto the nano LC-MS/MS system using a generic gradient method. During this screening, the retention time, peak width and limit of detection were determined. Further optimisation of the LC method is necessary for ghrelin and dynorphin A(1-13). Galanin and neuropeptide Y exhibit good peak shapes when high concentrations are injected (nM range). However, at lower concentrations (pM range) these peptides could not be measured. The limit of detection for bradykinin, neuromedin B, neuromedin N and neurotensin was found to be 7, 23, 1.5 and 2.6 pM respectively.
Third, a screening of the peptides was performed in basal microdialysis samples from the hippocampus of the rat. Neurotensin and neuromedin N were detected in a hippocampal dialysate. These preliminary experiments show that, after further optimisation, the quantification of the selected neuropeptides with nano LC-MS/MS is feasible.


References
[1] Sperk G, Drexel M, Pirker S (2009) Neuronal plasticity in animal models and the epileptic human hippocampus. Epilepsia 50:29-31
[2] Hökfelt T, Bartfai T, Bloom F (2003) Neuropeptides: opportunities for drug discovery. Lancet Neurol 2:463-472
[3] Lanckmans K, Sarre S, Smolders I, Michotte Y (2008) Quantitative liquid chromatography/mass spectrometry for the analysis of microdialysates. Talanta 74: 458-469