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

doi:10.1007/s00216-021-03437-x

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

Anal Bioanal Chem. 2021 Aug;413(19):4815-4824. doi: 10.1007/s00216-021-03437-x. Epub 2021 Jun 8.

Authors

Kevin Jeanne Dit Fouque^#^{1

2}, Julian D Hegemann^#³, Miguel Santos-Fernandez¹, Tung T Le⁴, Mario Gomez-Hernandez¹, Wilfred A van der Donk⁴, Francisco Fernandez-Lima^{5

6}

Affiliations

¹ Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, 33199, USA.
² Biomolecular Science Institute, Florida International University, 11200 SW 8th Street, Miami, FL, 33199, USA.
³ Institute of Chemistry, Technische Universität Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
⁴ Department of Chemistry and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA.
⁵ Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, 33199, USA. fernandf@fiu.edu.
⁶ Biomolecular Science Institute, Florida International University, 11200 SW 8th Street, Miami, FL, 33199, USA. fernandf@fiu.edu.

^# Contributed equally.

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 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.

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

MeSH terms

Amino Acid Sequence
Ion Mobility Spectrometry*
Peptides / chemistry*
Protein Conformation
Tandem Mass Spectrometry*

Substances

Peptides

Abstract

MeSH terms

Substances

Grants and funding