Lasso Peptides vs. Branched-Cyclic Topoisomers (1) (1) (1) (1)

Signature of Mechanically Interlocked Lasso Peptides Using Tandem Mass Spectrometry and Trapped Ion Mobility Spectrometry – Mass Spectrometry Melanie Perez,1 Javier Moreno,1 Julian Hegemann,2 Severine Zirah,3 Kevin Jeanne Dit Fouque,1 and Francisco Fernandez-Lima.1 1Department of Chemistry and Biochemistry, Florida International University, Miami, FL.33199, USA 2Department of Chemistry, University of Illinois, Urbana-Champaign, IL 61801, USA. 3Muséum National d'Histoire Naturelle, Sorbonne Universités, Laboratoire MCAM, 75005 Paris, France. 1 Biological Activities Antimicrobial and Antiviral (VIH) Receptor Antagonists (Glucagon, Endothelin, Atrial Natriuretic Factor…) Enzyme Inhibitors (ARNp, Prolyl-endopeptidase, MLCK…) Lasso Peptides MccJ25 Mechanically Interlocked Topology Ribosomally synthesized and post-translationally modified peptides (RiPPs) N-terminal macrocycle crossed by the C-terminal part. Loop and C-terminal tail Lasso topology maintained by bulky residues and/or disulfide bonds 2 Escherichia coli AY25: MccJ25 1992 : R. N. Farías 20 amino acids Gel filtration 1999 : S. Rebuffat 21 amino acids Cyclic (head-to-tail): NMR 2003 : D. J. Craik Lasso topology NMR Develop analytical efficient methods to obtain structural signatures of the lasso topology. Analytical Challenge 3 Lasso topology Essential for biological activities Lasso structures inaccessible by synthetic methods Branched-cyclic topology Unthreading of the lasso topology Produced by synthetic methods or by heating lasso peptides Mostly biologically inactive Lasso Branched-cyclic Need to characterize unambiguously lasso and branched-cyclic topologies (drug design) Issues 4 Nuclear Magnetic Resonance (NMR) Characterization of the tridimensional structures Identification of residues involved in the N-terminal macrocycle Enzymatic digestion + Chromatography Lasso structure resistant to carboxypeptidase Y Branched-cyclic structure digested by carboxypeptidase Y R. Ducasse et al. ChemBioChem, 2012, 13(3), 371-380. X. Xie et al. ChemBioChem, 2012, 13(5), 621-625. Limitations Large sample needed Purity Time-consuming Characterization Methods 5 Strategy Mass Spectrometry Mass measurements are not able to distinguish species with the same m/z (Topoisomers) Thermal Stability 20?C - 95?C (increment of 15?C) Proteolytic Stability (Carboxypeptidase Y) Localize the plug responsible for the stabilization of the lasso topology ? ? Second dimension of separation Tandem Mass Spectrometry (MS/MS) Collision Induced Dissociation (CID) Electron Capture Dissociation (ECD) Trapped Ion Mobility Spectrometry (TIMS-MS) Gas-phase identification and separation of the two topoisomers 6 Tandem Mass Spectrometry Collision Induced Dissociation (CID) Collision with neutral gas molecule (N2) Peptide bond cleavages: b and y ions Introduction of low-energy electrons c1? z2• c2? z1• b1 y2 b2 y1 CID ECD • z• c? [M+nH]n+ [M+nH](n-1)+• Electron Capture N-C? bond cleavages: c? and z• ions • Electron Capture Dissociation (ECD) 7 Collision Induced Dissociation (CID) MccJ25: Interlocked species with bi and yj fragments associated through the steric hindrance provided by the side-chain of the bulky residues No lasso-specific fragments S. Zirah et al. J. Am. Soc. Mass Spectrom. 2012, 22(3), 467-479. Lasso and branched-cyclic topologies are not clearly differentiated by CID 8 Electron Capture Dissociation (ECD) Different extent of hydrogen migration yielding c• / z? ions Lasso and branched-cyclic topologies are differentiated by ECD ECD does not allow to clearly discriminate both topologies in mixture. 9 10 CCS (Ų) E Trapped Ion Mobility Spectrometry – Mass Spectrometry (TIMS-MS) Holding the ions stationary in a flowing buffer gas by an electric field gradient Collision Cross Section (CCS): conformation of ions in the gas phase TIMS-MS results: Syanodin I Thermal Stability: 20?C - 95?C (increment of 15?C) 1h 1h 4h 4h After 4h: Lasso topology completely unthreaded at 95?C Lasso topology starts to unthread at 95?C (1h) Not thermally stable 12 TIMS-MS results: Syanodin I No separation between lasso and branched-cyclic peptides for the doubly protonated species Metal-Cationized Species permitted to clearly distinguish the two topologies 11 Ca2+ Cs+ Na+ Lasso Lcm [M+Ca]2+ [M+2Cs]2+ [M+H+Na]2+ Development of new multi-dimensional approaches 13 Conclusion Tandem Mass Spectrometry (MS/MS) Electron Capture Dissociation: Different extent of hydrogen migration yielding c• / z? ions Trapped Ion Mobility Spectrometry (TIMS) Metal-cationized species responsible for the topoisomer separation Not thermically stable: unthreads at 95?C Ion Mobility – Mass Spectrometry constitutes a fast and efficient method to unambiguously discern mechanically interlocked peptides from their unthreaded topoisomers Group Members Mentor Dr. Francisco Fernandez-Lima Post-doc David Butcher Kevin Jeanne Dit Fouque Graduate Students Alan McKenzie Alyssa Garabedian Anthony Castellanos Camilo Molano-Arevalo Jacob Porter Kendra Adams Paolo Benigni Collaborators Fundings R00GM106414 CHE-1654274 Acknowledgments Julian Hegemann Severine Zirah Undergraduate Students Javier Moreno 14