Journal
JOURNAL OF PROTEOME RESEARCH
Volume 19, Issue 2, Pages 634-643Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.9b00525
Keywords
glycosylation; neuropeptide; EThcD; crustacean nervous system; LC-MS/MS
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Funding
- National Science Foundation (NSF) [CHE-1710140]
- National Institutes of Health [R01 DK071801, U01 CA231081]
- NIH [NIH-NCRR S10RR029531]
- Wisconsin Alumni Research Foundation
- University of Wisconsin -Madison School of Pharmacy
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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 methods that enable deciphering this intricate low-abundance post-translational modification, 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 nonglycosylated peptides. Overall, 4 N-linked and 14 O-linked glycosylated neuropeptides have been identified for the first time in the crustacean nervous system. In addition, 91 novel putative neuropeptides have been discovered based on the collected HCD scans. This hybrid approach, coupling a shotgun method for neuropeptide discovery and targeted strategy for glycosylation characterization, enables the first report on glycosylated neuropeptides in crustaceans and the discovery of additional neuropeptides simultaneously. The elucidation of novel glycosylated neuropeptides sheds light on the crustacean peptidome and offers novel insights into future neuropeptide functional studies.
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