Czech Society for Mass Spectrometry

(13^th Czech Mass Spectrometry Conference and 11^th Informal Proteomic Meeting - WeO-06)
Breaking the Unbreakable: Structural Elucidation of the Hypermodified RiPP Nostatin A by IRMPD Mass Spectrometry

Jan Hájek ^1 *

1. Mikrobiologický ústav AVČR, centrum Algatech Třeboň

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a diverse and biologically significant class of natural products. However, their extensive post-translational modifications often pose major challenges for structural elucidation. Nostatin A (NosA, ~2.5 kDa) is a highly modified RiPP belonging to the nitrile hydratase–like leader peptide family (proteusins). Its 30-amino-acid core undergoes extensive structural modification, including the formation of nine thiazole/oxazole rings, eight dehydroalanine/dehydrothreonine residues, and an additional unique sactipeptide bridge. These modifications result in an extremely rigid and compact structure that resists conventional ESI ionization and collision-induced dissociation. Furthermore, NMR spectroscopy failed to provide sufficient data for complete structural elucidation. To overcome these limitations, infrared multiphoton dissociation (IRMPD) spectroscopy was employed, enabling controlled fragmentation and the observation of interpretable fragments across the m/z range of 200–2000. Interestingly, NosA predominantly fragmented into stable tripeptidic subunits composed of amino acid–oxa/thiazole–Dha/Dhb motifs, which resisted further cleavage. A novel approach combining empirical formula analysis of these smaller fragments allowed the determination of the complete molecular formula of Nostatin A.

* Corresponding author: zkoumi@gmail.com

References

1. Delawská, K., Hájek, J., Voráčová, K., Kuzma, M., Mareš, J., Vicková, K., Kádek, A., Tučková, D., Gallob, F., Divoká, P., Moos, M., Opekar, S., Koch, L., Saurav, K., Sedlák, D., Novák, P., Urajová, P., Dean, J., Gažák, R., . . . Hrouzek, P. (2024). Discov

Acknowledgement:

The authors gratefully acknowledge Alan Kádek and the BIOCEV research center for performing the IRMPD measurements on the SolariX FT-ICR mass spectrometer