4.7 Article

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

Journal

ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 413, Issue 19, Pages 4815-4824

Publisher

SPRINGER HEIDELBERG
DOI: 10.1007/s00216-021-03437-x

Keywords

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

Funding

  1. National Science Foundation Division of Chemistry, under CAREER award [CHE-1654274]
  2. Division of Molecular and Cellular Biosciences
  3. National Institutes of Health [R37 GM058822]

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This study investigated the structural signatures of the non-overlapping thioether ring pattern of the lanthipeptide Pcn2.8 using different mass spectrometry techniques. The results revealed the unique thioether ring pattern of Pcn2.8, providing a foundation for further understanding the structural elements of this peptide class.
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 alpha v beta 3 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 b(i) and y(j) 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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