Signature-Ion Triggered Mass Spectrometry Approach Enabled Discovery of N- and O-linked Glycosylated Neuropeptides in the Crustacean Nervous System.

Crustaceans are commonly used model organisms to study neuromodulation. Despite numerous reported crustacean neuropeptide families and their functions, there has been no report on neuropeptide glycosylation. This is in part due to a lack of sensitive method that enables deciphering this intricate low-abundance post-translational modification (PTM), even though glycosylation has been shown to play an important role in neuromodulation. Here, we describe the discovery of glycosylated neuropeptides with an enrichment-free approach taking advantage of signature oxonium ions produced in higher-energy collision dissociation (HCD) MS/MS spectra. The detection of the oxonium ions in the HCD scans suggests glycan attachment to peptides, allowing electron-transfer/higher-energy collision dissociation (EThcD) to be performed to selectively elucidate structural information of glycosylated neuropeptides that are buried in non-glycosylated peptides. Overall, 4 N-linked and 14 O-linked glycosylated neuropeptides have bee...