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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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Dual cationic–anionic profiling of metabolites in a single identified cell in a live Xenopus laevis embryo by microprobe CE-ESI-MS
In situ capillary microsampling with capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry (MS) enabled the characterization of cationic metabolites in single cells in complex tissues and organisms. For deeper coverage of the metabolome and metabolic networks, analytical approaches are needed that provide complementary detection for anionic metabolites, ideally using the same instrumentation. Described here is one such approach that enables sequential cationic and anionic (dual) analysis of metabolites in the same identified cell in a live vertebrate embryo. A calibrated volume was microaspirated from the animal-ventral cell in a live 8-cell embryo of Xenopus laevis , and cationic and anionic metabolites were one-pot microextracted from the aspirate, followed by CE-ESI-MS analysis of the same extract. A laboratory-built CE-ESI interface was reconfigured to enable dual cationic–anionic analysis with ∼5–10 nM (50–100 amol) lower limit of detection and a capability for quantification. To provide robust separation and efficient ion generation, the CE-ESI interface was enclosed in a nitrogen gas filled chamber, and the operational parameters were optimized for the cone-jet spraying regime in both the positive and negative ion mode. A total of ∼250 cationic and ∼200 anionic molecular features were detected from the cell between m / z 50–550, including 60 and 24 identified metabolites, respectively. With only 11 metabolites identified mutually, the duplexed approach yielded complementary information on metabolites produced in the cell, which in turn deepened network coverage for several metabolic pathways. With scalability to smaller cells and adaptability to other types of tissues and organisms, dual cationic–anionic detection with in situ microprobe CE-ESI-MS opens a door to better understand cell metabolism.
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- Award ID(s):
- 1832968
- PAR ID:
- 10098912
- Date Published:
- Journal Name:
- The Analyst
- Volume:
- 144
- Issue:
- 3
- ISSN:
- 0003-2654
- Page Range / eLocation ID:
- 892 to 900
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/C8AN01999A
