Discovery and De Novo Sequencing of Insect Neuropeptides Using the LTQ Orbitrap XL

Importance of the Topic

Neuropeptides are vital signaling molecules that regulate a wide array of insect physiological processes, including neurotransmission, hormone release, and muscle contraction. Detailed structural characterization of these peptides is essential for understanding their biological roles and for identifying new bioactive leads for pharmaceutical development.

Objectives and Study Overview

This work aimed to establish a robust, high‐throughput workflow for de novo sequencing of insect neuropeptides using nano LC‐MS/MS on an LTQ Orbitrap XL equipped with Higher Energy Collisional Dissociation (HCD). The study focused on neuroendocrine extracts from the corpora cardiaca/corpora allata complex of Periplaneta americana and P. australasiae to demonstrate comprehensive peptide identification and sequencing.

Methodology and Instrumentation

Adult cockroach neuropeptides were extracted in 50% methanol/0.1% formic acid, diluted, and injected into a 75 µm ID, 15 cm C18 nano‐HPLC column. Data were acquired on an LTQ Orbitrap XL in data‐dependent mode, performing both CID in the linear trap and HCD in the octapole collision cell. Full MS scans were recorded at 60,000 resolution, with 3 ppm precursor and 0.05 Da fragment tolerances. Automated de novo sequencing, database and error‐tolerant searches were carried out using PEAKS Studio 4.5 software; MASCOT searches were performed against SwissProt and nrNCBI.

Main Results and Discussion

High mass accuracy (<3 ppm) and the combination of CID and HCD fragmentation enabled near‐complete sequence coverage, including low‐mass immonium ions critical for distinguishing isobaric residues and post‐translational modifications. HCD spectra provided direct sequence ions for sulfated peptides that CID alone failed to resolve. Database matching confirmed over 20 known neuropeptides across families such as sulfakinins, pyrokinins, allatostatins, hypertrehalosaemic hormones, and corazonin. De novo scores exceeded 50% for most HCD spectra, placing correct sequences within the top five candidates. Homology searches using SPIDER on de novo results uncovered 21 additional putative neuropeptides, including a novel cockroach peptide DPGWNNLKGLWamide homologous to a mosquito allatostatin.

Benefits and Practical Applications

The presented workflow delivers a fully automated, sensitive approach for large‐scale discovery and sequencing of neuropeptides, overcoming limitations of traditional QTOF and MALDI‐MS/MS methods. It simplifies identification of modified and low‐abundance peptides, supporting functional studies in neurobiology and accelerating the search for insecticidal or therapeutic peptide leads.

Future Trends and Opportunities

Further enhancements may include optimized collision energy profiling, base peak ejection to unmask low‐intensity ions, and targeted MS^n experiments for ambiguous sequences. Expansion to other insect species and tissue types will broaden the neuropeptidomic landscape and reveal novel regulatory peptides.

Conclusion

The integration of HCD on the LTQ Orbitrap XL with automated de novo sequencing software constitutes a powerful platform for in‐depth neuropeptide analysis. This strategy achieves high sequence fidelity, robust identification of post‐translational modifications, and enables discovery of novel peptides in complex biological matrices.

References

1. Verhaert PD, Prieto-Conaway MC, Pekar TM, Miller K. Neuropeptide imaging on an LTQ with vMALDI source: The complete ‘all-in-one’ peptidome analysis. Int J Mass Spectrom. 2007;260:177–184.