1. INFRARED MULTIPLE PHOTON DISSOCIATION (IRMPD) SPECTROSCOPY OF b 7 IONS FROM MODEL ACETYLATED PEPTIDES Ahmet E. Atik and 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.  Direct absorption measurements and obtaining IR spectra of trapped ions challenging due to their extremly low densities (< 10 8 cm -3 )  IRMPD Spectroscopy of ions overcome this sensitivity problem Why IRMPD Spectroscopy for Trapped Ions

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

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

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

7. 7 oxazolone Diketopiperazine b 2 ? acylium H+H+ H+H+ H+H+

8. b n + (n = 2 - 4) ions protonated oxazolone structure 3, 4 [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. - H 2 O via nucleophilic attack from a backbone carbonyl oxygen to a carbonyl carbon An Oxazolone Structure b2b2 b3b3

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 YAGFLV oxa AGFLVY oxa GFLVYA oxa FLVYAG oxa LVYAGF oxa VYAGFL oxa internal amino acid eliminations (non-direct sequence b ions) were appeared

12. Macrocyclization of b Ions head-to-tail cyclization 5-7 YAGFLV oxa AGFLVY oxa GFLVYA oxa FLVYAG oxa LVYAGF oxa VYAGFL oxa 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. YAGFLV oxa b n + ions (n= 5, 6, 7 …)

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

14. N-terminal acetylation blocks the cyclization reaction and eliminates non-direct sequence fragment ions 7, 8 Macrocyclization of b Ions [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-K YAGFLV oxa

17. IRMPD SPECTROSCOPY OF b 7 IONS FROM MODEL ACETYLATED PEPTIDES Aim : to differentiate the macrocyclic structures of b 7 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 b 7 IONS FROM MODEL ACETYLATED PEPTIDES Comparison of IRMPD spectra of b 7 ions derived from :  K YAGFLV-NH 2 (no acetylation),  K Ac YAGFLVG (lysine side-chain is acetylated, ε-amine),  Ac-K YAGFLVG (N-terminal is acetylated, α-amine) characteristic macrocyclic absorption bands over 1000-2000 cm -1 range??? As a control experiment, Ac-K Ac YAGFLVG (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  K YAGFLV-NH 2 : no acetylation  K Ac YAGFLVG : side-chain is acetylated  Ac-K YAGFLVG : N-terminal is acetylated  Ac-K Ac YAGFLVG : both N-terminal and side-chain are acetylated Y : Tyrosine A : AlanineG : Glycine the synthetic model peptides were obtained from GL Biochem Ltd. (Shanghai, China) dissolved in a MeOH to give a conc. of 10 −4 M F : PhenylalanineL : LeucineV : Valine K : LysineAc : Acetyl Group

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) Energy: 8 to 50 MeV Peak current: 100 A Macro-pulse: length 10 μs, repetition rate: 6.25-25 Hz Micro-pulse: length 10 ps FWHM, pulse spacing 16 ns Emittance (rms): 40 pi mm mrad Spectral range: 3 to 150 microns (for different e - energies) Max. average power: 1 W @ 16ns/25Hz Max. peak power: 100 MW in 1ps Laser pulse length: 0.5 to 6 ps (adjustable) Table 1. Main characteristics of the CLIO facility

23. Results Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b 7 Ions K YAGFLV oxa Scheme 2

24. Results Scheme 3 Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b 7 Ions Ac-K YAGFLV oxa K(Ac) YAGFLV oxa

25. Results Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b 7 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 b 7 ion, the IRMPD spectra were recorded in the mid-IR range 1000-2000 cm -1 Figure 3

26. Results Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b 7 Ions two main experimental bands ~1510 cm -1 ~1675 cm -1 N-H bending (amide II) C=O stretching (amide I)  No bands over 1800-1900 cm -1  absence of oxazolone structure  N-terminal amine (α-amine) is more nucleophilic than lysine side-chain amine (ε-amine) group Figure 4

27. Results Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b 7 Ions 1614 cm -1 A A B B C C Figure 5

28. Results Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b 7 Ions the breakdwon graphs were constructed for internal amino acids eliminations from b 7 ions eliminations are ~ 4 % eliminations are ~ 6 % Figure 6

29. Results Ac-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide) no macrocyclization An oxazolone band needs to be appeared in the IRMPD spectrum of b 7 Figure 7 only direct sequence b ions

30. Results Figure 8

31. Results Ac-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide) breakdown graph of b 7 ion was constructed in order to see the cascade b ion series b7  b6  b5  b4  b3  b2  b1b7  b6  b5  b4  b3  b2  b1 Figure 9 Figure 10

32. Results Ac-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide) The b 1 ion is dissociated to form product ions at m/z 185, 171, 126 and 84 either consecutive or competitive pathway?? Figure 11

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

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

35. Conclusion 1  small band at 1614 cm -1 might be a signature for the “side-to-tail” cyclization of b 7 ion of Ac-KYAGFLVG  N-terminal amine (α-amine) is more nucleophilic than lysine side-chain amine (ε-amine) group (confirmed by IRMPD  stable b 1 ion (oxazolone) in the fragmentation of b 7 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: FT-ICR Ion Trap

38. THANKS FOR YOUR ATTENTION …