Transcript TY_Ankara

INFRARED MULTIPLE PHOTON
DISSOCIATION (IRMPD) SPECTROSCOPY OF
b7 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
Why IRMPD Spectroscopy for Trapped 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
IRMPD Spectroscopy of Trapped Ions
Vibrational spectrum offers:
 structural information
 location of charge (proton)
 presence (or absence) of chemical moieties
 symmetry
 secondary structure of proteins
 hydrogen bonding interactions
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
IRMPD Spectra of mass-selected Ions
Nick C. Polfer, Chem. Soc. Rev., 40, 2211-2221, 2011
Nomenclature of Peptide Fragment Ions
b2+
a2+
y2+
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.
Diketopiperazine
oxazolone
H+
H+
b2 ?
b2 ?
acylium
b2 ?
7
An Oxazolone Structure
b3
- H2O
b2
bn+ (n = 2 - 4) ions protonated oxazolone structure 3, 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.
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
bn+ ions (n= 5, 6, 7 …)
YAGFLVoxa
head-to-tail cyclization
5-7
YAGFLVoxa
AGFLVYoxa
Ring opening
GFLVYAoxa
FLVYAGoxa
LVYAGFoxa
VYAGFLoxa
[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.
M. Tirado and N. C. Polfer, Angew. Chem. Ed., 51, 6436-6438, 2012
Macrocyclization of b Ions
N-terminal acetylation blocks the cyclization reaction and eliminates 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.
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 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)
IRMPD SPECTROSCOPY OF b7 IONS
FROM MODEL ACETYLATED PEPTIDES
Comparison 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
K : Lysine
Y : Tyrosine
Ac : Acetyl Group
A : Alanine
F : Phenylalanine
L : Leucine
G : Glycine
V : Valine
• the synthetic model peptides were obtained from GL Biochem Ltd. (Shanghai, China)
• dissolved in a MeOH to give a conc. of 10−4 M
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)
Table 1. Main characteristics of the CLIO facility
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)
Results
Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 Ions
KYAGFLVoxa
Scheme 2
Results
Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 Ions
K(Ac)YAGFLVoxa
Ac-KYAGFLVoxa
Scheme 3
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
Figure 3
Results
Comparison 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)
 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
Figure 4
Results
Comparison of “head-to-tail” and “side-to-tail” Macrocyclization Chemistry of b7 Ions
A
A
B
B
C
C
1614 cm-1
Figure 5
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 %
Figure 6
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
Figure 7
Results
Figure 8
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
Figure 10
Figure 9
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 competitive pathway??
Results
Ac-K(Ac)YAGFLVG (Doubly Acetylated Model Octapeptide)
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
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:
•Ion Trap
•FT-ICR
THANKS FOR YOUR ATTENTION …