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Neuropeptides are important signaling molecules responsible for a wide range of functions within the nervous and neuroendocrine system. However, they are difficult to study due to numerous challenges, most notably their large degree of variability and low abundance in vivo . As a result, effective separation methods with sensitive detection capabilities are necessary for profiling neuropeptides in tissue samples, particularly those of simplified model organisms such as crustaceans. In order to address these challenges, this study utilized a capillary electrophoresis (CE)-matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) platform, building upon our previous design for improved neuropeptidomic coverage. The capillary was coated with polyethylenimine (PEI) to reduce peptide adsorption and reverse the electroosmotic flow, and large volume sample stacking (LVSS) was used to load and pre-concentrate 1 μL of sample. The method demonstrated good reproducibility, with lower than 5% relative standard deviation for standards, and a limit of detection of approximately 100 pM for an allatostatin III peptide standard. The method was tested on brain and sinus gland (SG) tissue extracts and enabled detection of over 200 neuropeptides per run. When comparing the number detected in brain extracts in a direct spot, 60-second fractions, and 30-second fractions, the continuous trace collection afforded by the CE-MALDI-MSI platform yielded the largest number of detected neuropeptides. The method was compared to conventional LC-ESI-MS, and though the number of neuropeptides detected by LC-ESI-MS was slightly larger, the two methods were highly complementary, indicating the potential for the CE-MALDI-MSI method to uncover previously undetected neuropeptides in the crustacean nervous system. These results indicate the potential of CE-MALDI-MSI for routine use in neuropeptide research.
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Enrichment and fragmentation approaches for enhanced detection and characterization of endogenous glycosylated neuropeptides
Abstract Glycosylated neuropeptides were recently discovered in crustaceans, a model organism with a well‐characterized neuroendocrine system. Several workflows exist to characterize enzymatically digested peptides; however, the unique properties of endogenous neuropeptides require methods to be re‐evaluated. We investigate the use of hydrophilic interaction liquid chromatography (HILIC) enrichment and different fragmentation methods to further probe the expression of glycosylated neuropeptides inCallinectes sapidus. During the evaluation of HILIC, we observed the necessity of a less aqueous solvent for endogenous peptide samples. This modification enabled the number of detected neuropeptide glycoforms to increase almost two‐fold, from 18 to 36. Product ion‐triggered electron‐transfer/higher‐energy collision dissociation enabled the site‐specific detection of 55 intact N‐ and O‐linked glycoforms, while the faster stepped collision energy higher‐energy collisional dissociation resulted in detection of 25. Additionally, applying this workflow to five neuronal tissues enabled the characterization of 36 more glycoforms of known neuropeptides and 11 more glycoforms of nine putative novel neuropeptides. Overall, the database of glycosylated neuropeptides in crustaceans was largely expanded from 18 to 136 glycoforms of 40 neuropeptides from 10 neuropeptide families. Both macro‐ and micro‐heterogeneity were observed, demonstrating the chemical diversity of this simple invertebrate, establishing a framework to use crustacean to probe modulatory effects of glycosylation on neuropeptides.
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- Award ID(s):
- 2108223
- PAR ID:
- 10397586
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- PROTEOMICS
- Volume:
- 23
- Issue:
- 3-4
- ISSN:
- 1615-9853
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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