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

Abstract

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 lowabundance 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.

Peak Filtering, Peak Annotation, and Wildcard Search for Glycoproteomics

“…The work reported here suggests some subjects for future study in both glycobiology and glycoproteomics software. On the biological side, the work reported here found O-linked glycosylation on neuropeptides, similar to corresponding recent discoveries of O-linked glycosylation on human and mouse insulin [35] and O-linked glycsylation on crustacean neuropeptides [36]. In all cases, the glycosylated peptides are of low abundance relative to the wildtype peptide, so the glycosylated forms may simply be a harmless product of some nonspecificity-"leakage"-of GalNAc transferases, which initiate most O-linked glycosylation in secreted proteins.…”

In-depth Site-specific Analysis of N-glycoproteome in Human Cerebrospinal Fluid and Glycosylation Landscape Changes in Alzheimer's Disease

“…Thus, glycopeptide enrichment is a key step in the success of glycopeptide analysis in complex biological samples. Many enrichment strategies have been developed in recent years, including HILIC, titanium dioxide (TiO 2 ) affinity, lectin affinity enrichment, hydrazide, and boronic acid chemistry ( 11 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ). To further reduce sample complexity and improve glycoproteome coverage, off-line fractionation such as high-pH fractionation (HpH) is often utilized due to its ease of implementation and its compatibility with large amounts of starting material, which has also been shown highly orthogonal to the subsequent LC-MS/MS analysis with low-pH reversed-phase chromatography ( 44 ).…”

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

“…In addition, ETD and ECD methods often benefit from hybrid approaches that use supplemental activation either during or following the electron-driven dissociation event to promote more extensive fragmentation ( 480 , 481 ). These hybrid methods have been shown to improve both N- and O-glycopeptide characterization ( 320 , 482 , 483 , 484 , 485 , 486 , 487 , 488 ), but they are effectively required for site-specific analyses of O-glycopeptides ( 52 , 113 , 114 , 305 , 473 ). As these nuances between N- and O-glycopeptides continue to be investigated, new acquisitions schemes, such as data-independent acquisition methods ( 489 , 490 , 491 , 492 , 493 , 494 ), are beginning to emerge that have the potential to enable consistent and reproducible characterization of larger and larger subsets of the glycoproteome.…”