Signaling peptides regulate neuronal circuit activities and a wide range of physiological processes. Measuring the dynamic changes of these important chemical messengers under different physiological conditions enables uncovering potential functions of these signaling molecules and at the same time represents significant analytical challenges. Equipped with high-resolution accurate mass (HRAM) Orbitrap instrumentation coupled with various separation techniques and isotopic and isobaric labeling strategies, we aim to explore peptidomic changes in the regulation of feeding behavior. Both crustacean and mammalian model organisms have been utilized to develop analytical methodologies and investigate the role of neuropeptides play in the regulation of this essential physiological function. Mass spectrometric imaging technology and in vivo microdialysis sampling tools have been developed to follow neuropeptide distribution and secretion with unprecedented details. To directly monitor feeding-induced changes in neuropeptide expression levels within the nucleus accumbens (Acb), we employed a combination of cryostat dissection, heat stabilization, neuropeptide extraction and quantitative neuropeptidomics, in rats either anticipating food or having recently completed a meal. Over 300 feeding-related neuropeptides were identified in rat Acb. Feeding altered expression levels of multiple neuropeptides of different families, especially opioid peptides such as enkaphalins and dynorphin, and non-opioid peptides including ProSAAS, orexin, and neuropeptide Y. We further investigated the regulatory functions of novel non-opioid neuropeptides from the ProSAAS family by infusing identified ProSAAS neuropeptides directly into the rat Acb, and monitoring spontaneous feeding and exploratory-like activity. Finally, in-depth characterization of unique post-translational modifications of signaling peptides using advanced instrumentation and hybrid fragmentation techniques will be discussed.
Hosted by Facundo Fernandez