Neuroendocrine regulatory peptide (NERP) -1 and NERP-2 are novel amidated peptides identified by peptidomic analysis of culture supernatant of an endocrine-type tumor cells. NERPs are processed out from VGF, a polypeptide secreted through the regulated pathway from neurons and endocrine cells (J. Biol. Chem., 282, 26354-60, 2007). We have shown that NERPs dose-dependently suppress the arginine vasopressin (AVP) release induced by hypertonic NaCl or angiotensin II in vivo and in vitro in the rats.
The natural occurrence of NERPs was confirmed by studies using specific antibodies and radioimmunoassays (RIAs). Before investigating the biological function of NERPs, we here measured rat tissue levels of the NERPs by RIA. They were present in the gastrointestinal tract, adrenal gland, and thyroid gland, and particularly abundant in the brain and pituitary. Characterization of immunoreactive (ir-) materials in the brain with a conventional approach (liquid chromatography coupled with RIA) elucidated the endogenous presence of NERP-1, NERP-2 and big NERP-2 in rat brain extract. NERP-1 and NERP-2 were confirmed by mass analysis of the ir-materials on a surface-enhanced laser desorption ionization (SELDI) mass spectrometer as well. The molecular structure of big NERP-2 was deduced as NERP-1+NERP-2 including an intervening tri-peptide by SELDI analysis and immunological properties.
While peptidomics has largely relied on mass spectrometric techniques for endogenous peptide determination, larger peptides often escape mass spectrometry-based identification. In this study, we demonstrated the significance of determining endogenous peptide forms with conventional methods, following mass spectrometry studies. Especially, a specific antibody recognizing the identified peptide is a crucial tool for the follow-up analysis.
