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

Kevin Jeanne Dit Fouque, Julian D. Hegemann, Miguel Santos-Fernandez, Tung T. Le, Mario Gomez-Hernandez, Wilfred A. van der Donk, Francisco Fernandez-Lima

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)4815-4824
Number of pages10
JournalAnalytical and bioanalytical chemistry
Issue number19
StatePublished - Aug 2021


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

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry


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