INFRARED MULTIPLE PHOTON DISSOCIATION (IRMPD) SPECTROSCOPY OF

b7 IONS FROM MODEL ACETYLATED PEPTIDES

Ahmet E. Atik and Talat YalcinDepartment 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

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

Why IRMPD Spectroscopy for Trapped Ions

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:

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

Nick C. Polfer, Chem. Soc. Rev., 40, 2211-2221, 2011

IRMPD Spectra of mass-selected Ions

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+ y2

+a2+

7

oxazolone Diketopiperazine

b2 ?

b2 ?

b2 ?

acylium

H+ H+H+

bn+ (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.

- H2O

via nucleophilic attack from a backbone carbonyl oxygen to a carbonyl carbon

An Oxazolone Structure

b2

b3

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

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

Macrocyclization of b Ions

YAGFLVoxa

AGFLVYoxa

GFLVYAoxa

FLVYAGoxa

LVYAGFoxa

VYAGFLoxa

internal amino acid eliminations (non-direct sequence b ions) were appeared

Macrocyclization of b Ions

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.

YAGFLVoxa

bn+ ions (n= 5, 6, 7 …)

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

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.

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

Side-to-Tail Macrocyclization of b Ions

Ac-KYAGFLVoxa

IRMPD SPECTROSCOPY OF b7 IONS FROM MODEL ACETYLATED

PEPTIDESAim : 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)

IRMPD SPECTROSCOPY OF b7 IONS FROM MODEL ACETYLATED

PEPTIDESComparison of IRMPD spectra of b7 ions derived from :

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

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

Y : Tyrosine A : Alanine G : 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 : Phenylalanine L : Leucine V : Valine

K : Lysine Ac : Acetyl Group

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

Centre Laser Infrarouge d’Orsay (CLIO)

Figure 2. Layout of the CLIO FEL (reprinted from http://clio.lcp.u-psud.fr/)

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

ResultsComparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 Ions

KYAGFLVoxa

Scheme 2

Results

Scheme 3

Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 Ions

Ac-KYAGFLVoxaK(Ac)YAGFLVoxa

ResultsComparison 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

Figure 3

ResultsComparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 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

ResultsComparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 Ions

1614 cm-1

A

A

B

B

CC

Figure 5

ResultsComparison 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 ~ 4 % eliminations are ~ 6 %

Figure 6

ResultsAc-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide)

no macrocyclization

An oxazolone band needs to be appeared in the IRMPD

spectrum of b7

Figure 7

only direct sequence b ions

Results

Figure 8

ResultsAc-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

Figure 9

Figure 10

ResultsAc-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide)

The b1 ion is dissociated to form product ions at m/z 185, 171, 126 and 84 either consecutive or competitive pathway??

Figure 11

ResultsAc-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide)

ResultsAc-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide)

m/z 185 and 126 have the same profile (competitive fragmentation pathway)

Figure 12

Scheme 5

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)

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

Mass Spectrometry systems:

•FT-ICR •Ion Trap

THANKS FOR YOUR ATTENTION …