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Ahmet E . Atik and T alat Yalcin

INFRARED MULTIPLE PHOTON DISSOCIATION (IRMPD) SPECTROSCOPY OF b 7 IONS FROM MODEL ACETYLATED PEPTIDES. Ahmet E . Atik and T alat Yalcin Department of Chemistry, Faculty of Science, Izmir Institute of Technology, Urla-Izmir, Turkey Oscar Hernandez and P hilippe Maître

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Ahmet E . Atik and T alat Yalcin

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  1. INFRARED MULTIPLE PHOTON DISSOCIATION (IRMPD) SPECTROSCOPY OF b7 IONS FROM MODEL ACETYLATED PEPTIDES Ahmet E. Atikand Talat Yalcin Department of Chemistry, Faculty of Science, Izmir Institute of Technology, Urla-Izmir, Turkey Oscar Hernandez and Philippe Maître Laboratoire de Chimie Physique, Université Paris Sud, UMR8000 CNRS, Faculté des Sciences, Bât. 350, 91405 Orsay Cedex, France Ozgur Birer Department of Chemistry, Faculty of Science, Koc University, Istanbul, Turkey TAC Light Sources (SR&FEL) International Users’ Meeting October 5-7, 2013

  2. Why IRMPD Spectroscopy forTrapped Ions • Direct absorption measurements and obtaining IR spectra of trapped ions • challenging due to their extremly low densities (< 108 cm-3) • IRMPD Spectroscopy of ions overcome this sensitivity problem

  3. IRMPD Spectroscopy ofTrapped Ions Vibrational spectrum offers: • structural information • location of charge (proton) • presence (or absence) of chemical moieties • symmetry • secondary structure of proteins • hydrogen bonding interactions

  4. Infrared Multiple Photon Dissociation (IRMPD) Spectroscopy • trapped ions are irradiated • a photon is absorbed (wavelength of laser =trapped ion’s vibr. mode) • intramolecular vibrational redistribution (IVR) • internal energy of the ion increases  fragment by unimolecular dissociation

  5. IRMPD Spectra of mass-selected Ions Nick C. Polfer, Chem. Soc. Rev., 40, 2211-2221, 2011

  6. Nomenclature of Peptide Fragment Ions y2+ b2+ a2+ Under low-energy CID conditions, protonated peptides typically fragment via cleavage at amide bonds to give N-terminal b-ions anda-ions and C-terminal y-ions1, 2 [1] Roepstorff, P.; Fohlmann, J. Biomed. Mass Spectrom. 1984,11, 601. [2] Biemann, K. Biomed. Environ. Mass Spectrom. 1988,16, 99.

  7. Diketopiperazine oxazolone H+ H+ H+ b2 ? b2 ? acylium b2 ? 7

  8. An Oxazolone Structure b3 - H2O b2 bn+ (n = 2-4) ions protonated oxazolone structure3, 4 via nucleophilic attack from a backbone carbonyl oxygen to a carbonyl carbon [3] Yalcin, T.; Khouw, C., Csizmadia, I. G.; Peterson, M. R.; Harrison, A. G.J. Am. Soc. Mass Spectrom. 1995, 6, 1165. [4]Yalcin, T.; Csizmadia, I. G.; Peterson, M. R.; Harrison, A. G.J. Am. Soc. Mass Spectrom. 1996, 7, 233.

  9. V.H. Wysocki et al., JACS, 130, 17644-17645, 2008

  10. B. Paizs, et al., JACS, 129, 5887- 5897, 2007

  11. Macrocyclization of b Ions YAGFLVoxa AGFLVYoxa GFLVYAoxa FLVYAGoxa LVYAGFoxa VYAGFLoxa internal amino acid eliminations (non-direct sequence b ions) were appeared

  12. bn+ ions (n= 5, 6, 7 …) Macrocyclization of b Ions YAGFLVoxa head-to-tail cyclization 5-7 YAGFLVoxa AGFLVYoxa GFLVYAoxa FLVYAGoxa LVYAGFoxa VYAGFLoxa Ring opening [5] Harrison, A. G.; Young, A. B.; Bleiholder, C.; Suhai, S.; Paizs, B. J. Am. Chem. Soc. 2006,128,10364. [6] Jia, C.; Qi, W.; He, Z. J. Am. Soc. Mass Spectrom. 2007,18,663. [7] Bleiholder, C.; Osburn, S.; Williams, T. D.; Suhai, S.; Van Stipdonk, M.; Harrison, A. G.; Paizs, B. J. Am. Chem. Soc. 130, 2008, 17774.

  13. M. Tirado and N. C. Polfer, Angew. Chem. Ed., 51, 6436-6438, 2012

  14. Macrocyclization of b Ions N-terminal acetylation blocks the cyclization reactionandeliminates non-direct sequence fragment ions 7, 8 [7] Bleiholder, C.; Osburn, S.; Williams, T. D.; Suhai, S.; Van Stipdonk, M.; Harrison, A. G.;Paizs, B. J. Am. Chem. Soc. 2008, 130,17774. [8] Harrison, A. G. J. Am. Soc. Mass Spectrom. 2009,20,2248.

  15. M. Tirado and N. C. Polfer, Angew. Chem. Ed., 51, 6436-6438, 2012

  16. Side-to-Tail Macrocyclization of b Ions Ac-KYAGFLVoxa

  17. IRMPD SPECTROSCOPY OF b7 IONS FROM MODEL ACETYLATED PEPTIDES Aim: to differentiate the macrocyclic structures of b7 ions that are formed either by “head-to-tail” or “side-to-tail” pathway • to form a regular macrocyclic structure (head-to-tail cyclization), N-terminal amine group must attack to the oxazolone’s carbonyl carbon • the lysine side chain amine group may also attack to form macrocyclic structure in N-terminal acetylated peptides(side-to-tail cyclization)

  18. IRMPD SPECTROSCOPY OF b7 IONS FROM MODEL ACETYLATED PEPTIDES • Comparison of IRMPD spectra of b7 ions derivedfrom : • KYAGFLV-NH2 (no acetylation), • KAcYAGFLVG (lysine side-chain is acetylated, ε-amine), • Ac-KYAGFLVG (N-terminal is acetylated, α-amine) characteristic macrocyclic absorption bands over 1000-2000 cm-1 range??? As a control experiment,Ac-KAcYAGFLVG (doubly acetylated) peptide was used for obtaining IRMPD spectrum which needs to contain a characteristic band over 1800 cm-1 due to the N-protonated oxazolone structure

  19. Experimental • KYAGFLV-NH2: no acetylation • KAcYAGFLVG : side-chain is acetylated • Ac-KYAGFLVG : N-terminal is acetylated • Ac-KAcYAGFLVG : both N-terminal and side-chain are acetylated K : Lysine Ac : Acetyl Group Y : Tyrosine A : Alanine G : Glycine F : Phenylalanine L : Leucine V : Valine • thesynthetic model peptides were obtained from GL Biochem Ltd. (Shanghai, China) • dissolved in a MeOH to give a conc. of 10−4 M

  20. Experimental • For the FEL experiments: IRMPD spectroscopy experiments were performed at the Centre Laser Infrarouge d’Orsay (CLIO) FEL facility at the University of Paris-Sud XI in Orsay, France • FT-ICR MS with a 7 Tesla magnet (Apex Qe, Bruker Daltonics; Billerica, MA, USA) • Paul-type ion-trap (QIT) MS (Esquire 3000+, Bruker Daltonics; Bremen, Germany) • For the mass spectra and breakdown graphs: • LTQ XL linear ion-trap MS (Thermo Finnigan, San Jose, CA, USA) equipped with an ESI source was used • Q-TRAP, Applied Biosystems / MDS Sciex, Concord, Canada) equipped with a turbo ion spray source

  21. Centre Laser Infrarouge d’Orsay(CLIO) Figure 2. Layout of the CLIO FEL (reprinted from http://clio.lcp.u-psud.fr/)

  22. Centre Laser Infrarouge d’Orsay(CLIO) Table 1. Main characteristics of the CLIO facility

  23. Results Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 Ions KYAGFLVoxa Scheme 2

  24. Results Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 Ions K(Ac)YAGFLVoxa Ac-KYAGFLVoxa Scheme 3

  25. Results Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 Ions non-direct sequence b ions (internal amino acid losses) were appeared with different relative intensities in each mass spectra to clarify the gas-phase macrocyclic structure of each b7 ion, the IRMPD spectra were recorded in the mid-IR range 1000-2000 cm-1 Figure3

  26. Results Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 Ions two main experimental bands ~1675 cm-1 ~1510 cm-1 N-H bending (amide II) C=O stretching (amide I) • N-terminal amine (α-amine) is more nucleophilic than lysine side-chain amine (ε-amine)group • No bands over 1800-1900 cm-1  absence of oxazolone structure Figure4

  27. Results Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 Ions A A B B C C 1614 cm-1 Figure5

  28. Results Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 Ions the breakdwon graphs were constructed for internal amino acids eliminations from b7 ions eliminations are ~ 6 % eliminations are ~4 % Figure6

  29. Results Ac-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide) only direct sequence b ions no macrocyclization An oxazolone band needs to be appeared in the IRMPD spectrum of b7 Figure7

  30. Results Figure8

  31. Results Ac-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide) breakdown graph of b7 ion was constructed in order to see the cascade b ion series b7 b6  b5  b4  b3  b2  b1 Figure10 Figure 9

  32. Results Ac-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide) Figure 11 The b1 ion is dissociated to form product ions at m/z 185, 171, 126 and 84 either consecutive or competitivepathway??

  33. Results Ac-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide)

  34. Results Ac-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide) Figure 12 m/z 185 and 126 have the same profile (competitive fragmentation pathway) Scheme 5

  35. Conclusion 1 • small band at 1614 cm-1 might be a signature for the “side-to-tail” cyclization of b7 ion of Ac-KYAGFLVG • N-terminal amine (α-amine) is more nucleophilic than lysine side-chain amine (ε-amine) group (confirmed by IRMPD • stable b1 ion (oxazolone) in the fragmentation of b7 ion of Ac-K(Ac)YAGFLVG (confirmed by both its IRMPD and mass spectrum individually)

  36. Conclusion 2 • protein-peptide non-covalent complex • Chirality recognition • structural characterization • differentiate isomeric mixture of peptides/proteins • structure of neutral elimination in gas phase can be determined

  37. Mass Spectrometry systems: • Ion Trap • FT-ICR

  38. THANKS FOR YOUR ATTENTION …

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