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

Analytical and Bioanalytical Chemistry volume 413, pages 4815–4824 (2021)Cite this article

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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  1. Kevin Jeanne Dit Fouque and Julian D. Hegemann contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, 33199, USA

    Kevin Jeanne Dit Fouque, Miguel Santos-Fernandez, Mario Gomez-Hernandez & Francisco Fernandez-Lima

  2. Biomolecular Science Institute, Florida International University, 11200 SW 8th Street, Miami, FL, 33199, USA

    Kevin Jeanne Dit Fouque & Francisco Fernandez-Lima

  3. Institute of Chemistry, Technische Universität Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany

    Julian D. Hegemann

  4. Department of Chemistry and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA

    Tung T. Le & Wilfred A. van der Donk

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  1. Kevin Jeanne Dit Fouque
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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