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Exploring structural signatures of the lanthipeptide prochlorosin 2.8 using tandem mass spectrometry and trapped ion mobility-mass spectrometry

Abstract

Lanthipeptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by intramolecular thioether cross-links formed between a dehydrated serine/threonine (dSer/dThr) and a cysteine residue. Prochlorosin 2.8 (Pcn2.8) is a class II lanthipeptide that exhibits a non-overlapping thioether ring pattern, for which no biological activity has been reported yet. The variant Pcn2.8[16RGD] has been shown to bind tightly to the αvβ3 integrin receptor. In the present work, tandem mass spectrometry, using collision-induced dissociation (CID) and electron capture dissociation (ECD), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) were used to investigate structural signatures for the non-overlapping thioether ring pattern of Pcn2.8. CID experiments on Pcn2.8 yielded bi and yj fragments between the thioether cross-links, evidencing the presence of a non-overlapping thioether ring pattern. ECD experiments of Pcn2.8 showed a significant increase of hydrogen migration events near the residues involved in the thioether rings with a more pronounced effect at the dehydrated residues as compared to the cysteine residues. The high-resolution mobility analysis, aided by site-directed mutagenesis ([P8A], [P11A], [P12A], [P8A/P11A], [P8A/P12A], [P11A/P12A], and [P8A/P11A/P12A] variants), demonstrated that Pcn2.8 adopts cis/trans-conformations at Pro8, Pro11, and Pro12 residues. These observations were complementary to recent NMR findings, for which only the Pro8 residue was evidenced to adopt cis/trans-orientations. This study highlights the analytical power of the TIMS-MS/MS workflow for the structural characterization of lanthipeptides and could be a useful tool in our understanding of the biologically important structural elements that drive the thioether cyclization process.

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Funding

Financial support was from the National Science Foundation Division of Chemistry, under CAREER award CHE-1654274, with co-funding from the Division of Molecular and Cellular Biosciences to FFL and the National Institutes of Health (R37 GM058822) to WAV.

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KJDF and JDH contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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Correspondence to Francisco Fernandez-Lima.

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Jeanne Dit Fouque, K., Hegemann, J.D., Santos-Fernandez, M. et al. Exploring structural signatures of the lanthipeptide prochlorosin 2.8 using tandem mass spectrometry and trapped ion mobility-mass spectrometry. Anal Bioanal Chem 413, 4815–4824 (2021). https://doi.org/10.1007/s00216-021-03437-x

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Keywords

  • Pcn2.8
  • Lanthipeptide
  • Thioether cross-link
  • cis/trans-configuration
  • Collision-induced dissociation
  • Electron capture dissociation
  • Trapped ion mobility spectrometry