Enhanced Coverage of Insect Neuropeptides in Tissue Sections by an Optimized Mass Spectrometry Imaging Protocol

Significance of the topic

The study of insect neuropeptides is critical for understanding physiological regulation in insects and for developing novel pest control strategies. Mass spectrometry imaging offers comprehensive spatial mapping of neuropeptides, overcoming limitations of immunohistochemistry and enabling high-resolution analysis of molecular distributions in tissue sections.

Objectives and study overview

This study aims to optimize a MALDI mass spectrometry imaging protocol to achieve enhanced coverage and spatial resolution of insect neuropeptides in the retrocerebral complex of Periplaneta americana. The protocol enables near-complete detection of neuropeptides from single sections and allows reconstruction of RCC compartmentalization and differential peptide processing under various conditions.

Methodology and instruments used

• Sample preparation: RCC extraction, embedding in gelatin, cryo-sectioning at 14–20 µm, thaw mounting on ITO slides.
• Tissue treatment: Sequential washing in 70% and 100% ethanol, vacuum drying.
• Matrix application: 5 mg/ml CHCA in 50% acetonitrile with 2% TFA using SunCollect dispenser.
• MS acquisition: rapifleX MALDI-TOF in positive reflector mode, m/z range 600–3200, pixel size 15 µm, laser repetition 5 kHz.
• Data analysis: flexImaging, flexAnalysis and SCiLS Lab software with confirmation of neuropeptide identities from reference databases.

Main results and discussion

• Detection of over 100 peptides and approximately 60 bioactive neuropeptides from 15 precursor genes in a single RCC section.
• Distinct spatial distributions for corazonin, pyrokinins, allatostatin-A and allatotropin, matching known neurosecretory pathways.
• Spatial segmentation analysis reconstructed RCC compartments and distinguished glandular and neurohemal regions with high specificity.
• High mass resolution enabled discrimination of ions differing by 0.2 Da, such as pyrokinin-1 and sodium-adduct of adipokinetic hormone-2.

Benefits and practical applications

• Comprehensive neuropeptidome profiling in single tissue sections with reproducible high spatial resolution.
• Ability to map and quantify region-specific peptide processing and distribution in insect neuroendocrine tissues.
• Potential to study dynamic changes in the neuropeptidome in response to environmental stressors or insecticide exposure.
• Enhanced throughput for insect neuroscience, developmental biology and pest management research.

Future trends and potential applications

• Integration of MSI with other imaging modalities for multimodal analysis of insect tissues.
• Extension to other insect models to investigate developmental and adaptive neuropeptide dynamics.
• Application in screening novel bioactive compounds targeting specific neuropeptide pathways.
• Advancements in instrumentation to further improve spatial resolution and throughput.

Conclusion

The optimized rapifleX MALDI-TOF imaging protocol delivers near-complete coverage of insect neuropeptides in the retrocerebral complex with high spatial resolution and mass accuracy. This approach enables detailed compartmental mapping and discrimination of closely related peptides, supporting advanced studies in insect physiology and pest control research.

Reference

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