Project Summary/Abstract Neuropeptides (NPs), important neuromodulators and signaling molecules, are very diverse in structure and can have distinct functions altered by location, concentration, and co-modulation with other NPs, though the functions of many are unknown. Their global characterization is challenging due to their diversity and low abundance in vivo. Glycosylation is a complex post-translational modification that can further complicate NP characterization. Defined by the addition of a glycan comprised of various monosaccharides in different combinations and configurations, peptide and protein glycosylation can modify binding affinity, trafficking, lifetime, and function. Although glycan characterization is becoming more common, the functional role of glycosylation on NPs remains sparsely studied and unclear. Characterization is partially limited by its even lower abundance in vivo compared to non-modified NPs and thus requires new and effective strategy. This can be performed by mass spectrometry (MS), a sensitive and selective analytical technique, which is able to identify and localize various glycosylation modifications of NPs. While not previously studied in the context of NPs, hypoxia exposure has been shown to lead to dysregulation of glycosylation, both in environmental and tumor contexts. Recently, NPs have been implicated in stress responses to hypoxic conditions, though modified NPs have not been examined. The goal of this project is to address this critical knowledge gap and to elucidate the role of glycosylation on NPs due to exposure to hypoxic stress. This will be performed in the crustacean model organism due to its simple, well characterized neuroendocrine system and the homology present between some human NPs. Aim 1 focuses on developing a workflow for the increased detection and characterization of glycoNPs present in blue crabs, Callinectes sapidus. This will be performed by the enrichment of the low abundance glycosylated NPs prior to MS analysis, as well as the optimization of a MS fragmentation method. Aim 2 will implement the developed strategy from Aim 1 to characterize the hypoxia stress-induced modulation of glycans on NPs both quantitatively and qualitatively, looking at the relative changes in expression abundance and expressed glycoforms. Aim 3 will lead to the further elucidation of glycosylation roles and effects by studying structural trends through ion-mobility spectrometry MS, a technique that can measure structural information through gas-phase mobility values. Correlation between structural characteristics of glycoNPs versus non-glycosylated NPs can provide insight into their varying binding affinities and functional roles. Together, these aims will provide insight into the roles of glycosylation on NPs and how their regulation is modulated by biological coping mechanisms resulting from a hypoxic environment.