Structural Characterization of Cyclic Peptides using a Quadrupole Time-of-Flight Mass Spectrometer

Significance of the Topic

Cyclic peptides represent an emerging class of therapeutics with enhanced in vivo stability and cell permeability. Accurate structural characterization is essential for understanding their bioactivity and ensuring quality control during drug development.

Objectives and Study Overview

This work presents a comprehensive LC/Q-TOF MS method for the structural analysis of the cyclic peptide cyclosporin A. The goal was to achieve full sequence coverage and precise fragment assignment via high-resolution MS and MS/MS experiments.

Applied Methodology and Instrumentation

• UHPLC system: Nexera X2 with Shim-pack GISS column (2.1×50 mm, 1.9 µm)
• Mobile phases: 0.1 % formic acid in water (A) and in acetonitrile (B); gradient from 60 to 95 % B
• Flow rate: 0.3 mL/min; column temperature: 50 °C; injection volume: 1 µL
• Mass spectrometer: LCMS-9030 Q-TOF with >30 000 resolution (FWHM), <1 ppm mass accuracy, 100 Hz acquisition rate
• Data analysis: ACD/MS Workbook Suite for formula prediction and fragment assignment

Main Results and Discussion

Accurate mass measurement confirmed [M+H]+ at m/z 1202.8486 and [M+2H]2+ at m/z 601.9279 within ±1 ppm. MS/MS spectra provided fragmentation along a, b, c, x, y, and z series, with mass errors below 1 mDa. The combined analysis achieved 100 % coverage of the cyclosporin A structure. Supplementary MS3 experiments on an IT-TOF system corroborated the MS/MS assignments.

Benefits and Practical Applications

The method enables rapid and reliable sequencing of cyclic peptides, facilitating drug discovery, metabolite profiling, and quality assurance workflows. High mass accuracy simplifies spectral interpretation and reduces manual verification.

Future Trends and Potential Applications

Emerging directions include integration with ion mobility for conformer separation, automated spectral annotation using advanced informatics, and extension of the workflow to other macrocyclic compounds and complex peptidomimetics.

Conclusion

The developed LC/Q-TOF MS approach delivers comprehensive structural insights into cyclosporin A using only high-resolution MS/MS data. It demonstrates a robust platform for the accurate sequencing of cyclic peptides in research and industrial settings.

References

Matsubara T, Inohana Y, Hirano I Structural Characterization of Cyclic Peptides using a Quadrupole Time-of-Flight Mass Spectrometer ASMS 2019 PO-CON1892E