Analytic framework for peptidomics applied to large-scale neuropeptide identification

Large-scale mass spectrometry-based peptidomics for drug discovery is relatively unexplored because of challenges in peptide degradation and identification following tissue extraction. Here we present a streamlined analytical pipeline for large-scale peptidomics. We developed an optimized sample preparation protocol to achieve fast, reproducible and effective extraction of endogenous peptides from sub-dissected organs such as the brain, while diminishing unspecific protease activity. Each peptidome sample was analysed by high-resolution tandem mass spectrometry and the resulting data set was integrated with publically available databases. We developed and applied an algorithm that reduces the peptide complexity for identification of biologically relevant peptides. The developed pipeline was applied to rat hypothalamus and identifies thousands of neuropeptides and their post-translational modifications, which is combined in a resource format for visualization, qualitative and quantitative analyses

Characterization of murine melanocortin receptors mediating adipocyte lipolysis and examination of signalling pathways involved

International audienceThe melanocortin receptors (MCRs) belong to the G-protein coupled receptors (family A). So far, 5 different subtypes have been described (MC1R- MC5R) and of these MC2R and MC5R have been proposed to act directly in adipocytes and regulate lipolysis in rodents. Using ACTH and a-melanocyte stimulating hormone (a-MSH) generated from proopiomelanocortin (POMC), as well as synthetic MSHanalogues to stimulate lipolysis in murine 3T3-L1 adipocytes it is shown that MC2R and MC5R are lipolytic mediators in differentiated 3T3-L1 adipocytes. Involvement of cAMP, phosphorylated extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (PKB), adenosine 5' monophosphateactivated protein kinase (AMPK) and Jun-amino-terminal kinase (JNK) in MCR mediated lipolysis were studied. Interestingly, results obtained in 3T3-L1 cells suggest that lipolysis stimulated by a-MSH, NDPa- MSH, MT-II, SHU9119 and PG-901 is mediated through MC5R in a cAMP independent manner. Finally, we identify essential differences in MCR mediated lipolysis when using 3T3-L1 cells compared to primary adipocytes