ASMS Conference on Mass Spectrometry and Allied Topics edition:52 location:Nashville, Tennessee U.S.A. date:May 23-27, 2004
Single avian median eminence LC-MS neuropeptide profiling; putative new neuropeptides found
This investigation aimed at identifying new avian hypophysiotropic neuropeptides in the Japanese quail (Coturnix coturnix japonica). The pituitary is controlled by the hypothalamus, making the median eminence the ideal substrate for the discovery of concentrated hypophysiotropic neuropeptides, prior to their release and transport via the portal system towards their place of action, the pituitary. As the pituitary gland (hypophysis) is of paramount importance in the regulation of avian reproduction, thorough knowledge of neuropeptide hormones controlling and modulating pituitary neuropeptide release is imperative for contemporary animal physiologists.
A single female quail median eminence was collected and neuropeptides were extracted using an acidic methanol extraction (sonication in 90% methanol, 1% formic acid for 1h). The extract was analysed using nanoscale HPLC (LC Packings Ultimate) coupled to a Q-TOF mass spectrometer (Micromass). [M+2H]2+, [M+3H]3+ and [M+4H]4+ components were automatically selected and fragmented using predefined collision energy profiles. Peak lists were extracted from the LC-MS data files using the ProteinLynx data processing routine. For manual processing, the MS/MS chromatograms were screened for suitable peptide MS/MS spectra, which were deconvoluted and deisotoped (MaxEnt 3). De novo sequencing was done in PepSeq.
Mascot (Matrix Science) searching with the LC-MS/MS peak lists was done using the default parameters, except for taxonomy (Chordata), enzyme (none), instrument (ESI-QUAD-TOF) and using the following variable modifications: amide (C-term), oxidation (M) and pyro-Glu (N-term Q). Mascot searching identified peptides derived from the gonadotropin inhibitory hormone (GnIH) precursor, and peptides derived from pro-enkephalin A. Also, the neurokinin B neuropeptide was found. One of the pro-enkephalin A-derived peptides, although not previously described as an active peptide, shows high homology with amphibian and mammalian sequences. Future research will point out if this is a conserved neuropeptide. An almost identical result was obtained with manual processing and de novo sequencing, except for one peptide which could not be fully de novo sequenced to the C-terminal end, but which bears resemblance to an EST, identified as chicken chromogranin B. An alignment (at the region of interest) of mammalian CgB with partial putative chicken CgB and the quail partial peptide shows two adjacent conserved dibasic cleavage sites, confirming its putative neuropeptide character. For a number of MS/MS spectra, only short sequence tags (2-6 amino acids) were derived that could not be identified with currently available database searching.