Elucidating the Neuropeptidome Implicated in Crustacean Feeding Processes through Multiplexed Data-Independent Acquisition Mass Spectrometry - Project Summary/Abstract Feeding processes are intertwined in a number of human health investigations, though the precise mechanisms through which these operate remains elusive. Neuropeptides are signaling molecules that provide a dynamic fingerprint of neuronal function, serving as a rich resource for pathway elucidation and biomarker discovery. Despite this potential, neuropeptide analyses are met with many hurdles, stemming from their low in vivo abundance and a lack of computational resources for confident identification and accurate quantification. It is also imperative that the investigation of feeding processes through neuropeptides is conducted in a global and unbiased manner, as neuropeptide activity is heavily co-modulated by other neuropeptides. My proposed aims to bridge these gaps to elucidate feeding mechanisms through holistic investigation of the neuropeptidome. This task will be accomplished through use of a crustacean model organism, Cancer borealis, revered as the premier model for neuroendocrine investigations of this nature for its simpler, yet well-characterized, nervous system with established neuropeptide homology to humans. A multipronged approach through strategic employment of data-independent acquisition (DIA) mass spectrometry, key for overcoming the inherent bias of the mass spectrometer toward highly abundant analytes, will be used to profile and subsequently quantify neuropeptides from C. borealis neuroendocrine tissue extract. As neuropeptide identifications are not immediately amenable to existing software packages designed for protein analysis, partially due to the endogenous nature in which they are analyzed, a software suite, named EndoGenius, will be designed and optimized specifically for identification and quantification of neuropeptides obtained from DIA experiments (Aim 1). In parallel, a spectral library of crustacean neuropeptides will be built for identification of neuropeptides from DIA experiments, a strategy to maximize the extent of neuropeptide elucidation and overall coverage (Aim 2). To improve resolution of neuropeptide abundance alterations and enable multiplexed analysis, isobaric tagging of neuropeptides will be utilized together with DIA mass spectrometry, providing a complete picture of neuropeptide profile changes in response to feeding (Aim 3). Each aim is designed for specific application to feeding samples, collected at four time points, contributing to the overall neuropeptidome associated with these critical behavioral functions. I hypothesize that these methods will comprehensively resolve the temporal profile of neuropeptidome changes induced following feeding activity with high levels of quantitative accuracy. Following these advancements, the findings can be used to elucidate feeding regulatory mechanisms as well as neuropeptide biomarkers that are associated with feeding disorders.
