Characterization of O-Linked Glycosylated Neuropeptides in the American Lobster (Homarus americanus): The Use of Peptide Labeling Following Beta Elimination

Neuropeptides are a class of small peptides that govern various neurological functions, and the American lobster (Homarus americanus) provides a model system for their characterization. Neuropeptides are commonly post-translationally modified (PTM), and one common PTM is glycosylation. Past research in the Stemmler lab has found glycosylated neuropeptides in H. americanus; however, the extent and biological role of this modification has not been well characterized. This study was undertaken to determine the number of glycosylated peptides in the sinus glands of H. americanus and to develop an approach to tag the site of glycosylation using beta-elimination chemistry. LC-MS paired with high pH reverse phase fractionation was used to survey for glycosylated neuropeptides and beta elimination with an amine tag was used as an approach to characterize the site of glycosylation. Our results indicate that high pH fractionation is a useful approach to simplify complex mixtures of neuropeptides and improve glycopeptide detection. Efforts to use beta elimination and tagging to characterize glycosylated neuropeptides have been less successful. Beta elimination of full length peptides resulted in peptide degradation. An approach utilizing chymotrypsin to reduce peptide size coupled with beta elimination and labeling with 2-dimethylaminoethanethiol showed less evidence for degradation, and this approach yielded data isolating two potential serine residues for the site of glycosylation; however, the data was not sufficient to distinguish the two sites. Work to optimize reaction conditions using a glycopeptide standard showed that multiple isomeric products were formed during beta elimination. With the goal of optimizing reaction conditions, future work will further examine reaction kinetics to eventually apply the approach to the entire sinus gland