1. IR ion spectroscopy at FELIX
Peptide dissociation and analytical applications
Anouk Rijs
Jos Oomens
Radboud University – FELIX laboratory – Nijmegen – The Netherlands
© M. Molle

2. Ion spectroscopy: photo-dissociation as spectroscopic tool
Mass spectrum
IR spectrum

3. Ion spectroscopy: photo-dissociation as spectroscopic tool
J. Am Chem. Soc. 1981

4. FELIX - Free Electron Laser
Int’l user facility
undulator magnets
IR-beam
Wavelength tunable 3 – 150 mm
Pulse energy <100 mJ per 5 ms pulse
FWHM bandwidth >0.4% of l
International user facility
gun
e-beam
accelerator
mirror

5. Ion spectroscopy: photo-dissociation as spectroscopic tool
Polfer, Oomens, PCCP 2007, 9, 3804

6. Application of ion spectroscopy: understanding peptide fragmentation
collisional
activation
mobile
proton
nucleophilic attack
amide bond cleavage
b/y
fragments

7. Application of ion spectroscopy: understanding peptide fragmentation

8. Experimental: modified Bruker Amazon Speed ETD
1.) Mention concentrations used when describing ESI source
2.) Glass transfer capillary
3.) dual ion funnel, off axis to each other, increases sensitivity 1-2 orders of magnitude

9. Experimental: modified Bruker Amazon Speed ETD

10. Experimental: modified Bruker Amazon Speed ETD
From FEL
To photodiode

11. Experimental: modified Bruker Amazon Speed ETD
Parent - pTyrH+
Fragment - pTyrH+
CO2 laser
Near complete dissociation
 Favourable overlap ion cloud / laser focus (~1 mm)

12. Comparison ion trap vs. FT-ICR
Amazon
Pro3H+

13. Bruker Amazon: advantages over our FTMS
High sensitivity: large dynamic range  low abundance species
High sensitivity: multiple MSn stages  follow rxn network
High duty cycle: reduced time per IR spectrum
ETD option: study ETD reaction products (and other radical ions, charge-reduced product ions)

14. Deamidation of Gln and Asn containing peptides
A seemingly simple reaction …
NH3 neutral loss followed by nucleophilic attack

15. Deamidation of protonated Asn
Heaton, Armentrout, JASMS 2009, 20, 852

16. Deamidation of protonated Gln and Asn
Kempkes, Martens, Grzetic, Berden, Oomens, Rapid Comm Mass Spectrom 2016, 30, 483

17. Following the reaction network Deamidation of AlaGln H+
Possible leaving groups
Possible nucleophiles

18. Four dipeptides: AlaAsn, AsnAla AlaGln, GlnAla
N-terminus or side chain?
Cyclization or not?
Which nucleophile?
Protonation site?
Only rxns leading to 5- or 6-membered ring products are indicated.

19. Following the reaction network: Deamidation of GlnAlaH+
MS/MS
MS/MS/MS
- 17
- 18

20. Following the reaction network: Deamidation of AsnAlaH+
MS/MS
MS/MS/MS
- 17
- 17

21. Following the reaction network: deamidation of AsnAlaH+
MS/MS
MS/MS/MS

22. Following the reaction network: deamidation of AsnAlaH+

23. Deamidation of protonated AsnAla, AlaAsn, GlnAla, AlaGln
NH3-loss from side chain
5-membered rings formed
nucleophile analogous to AA
secondary NH3 or H2O loss
MS3 fragments characterized
consistent rxn network

24. Mapping the entire reaction network
Lisanne Kempkes

25. electron transfer dissociation
Spectroscopic investigation of ETD product ions [M + nH]n+ + A [M + nH](n-1)+ + A ET
fluoranthene

26. ETD as alternative for CID in peptide sequencing
Coon, AC 2009, 81, 3208

27. Electron Transfer Dissociation (ETD)
N-Ca cleavage: c and z• ions
open shell fragments!

28. Model Peptide - [AAHAR + 2H]2+
ExD of AAXAR studied in detail by Turecek
MSn and computational
Validated computational methods, low energy structures, fragmentation pathways, radical migration reactions, etc.
Turecek, Chung, Moss, Wyer, Ehlerding, Holm, Zettergen, Brondsted Nielsen, Hvelpund, Chamot-Rooke, Bythell, Paizs, JACS 2010, 132, 10728
Tureček, Moss, Chung, IJMS, 2012, 330–332, 207
Ledvina, Chung, Hui, Coon, Turecek, JASMS 2012, 23, 1351

29. z1•+ fragment: an a-radical. .
H-atom transfer
d to a-carbon
TS at 115 kJmol-1

30. z2•+: IR spectrum identifies a-radical
a-radical lower in energy than d-radical
Martens, Grzetic, Berden, Oomens, Nature Commun. 2016, 7, 11754

31. z3•+: a-radical a-radical a-radical, His tautomer b-radical
Lower in energy
b-radical
Martens, Grzetic, Berden, Oomens, Nature Commun. 2016, 7, 11754

32. First IR spectra of ETD product ions
a-radicals dominate: no H-atom transfer
Martens, Grzetic, Berden, Oomens, Nature Commun. 2016, 7, 11754

33. Mass spectrometry as analytical tool
metabolites
environmental
food analysis
petreolomics
illicit drug testing
airport security
proteomics
glycomics
Mass spectrometry as analytical tool
high sensitivity
extremely high resolution
A.G. Marshall and coworkers, NHFML, Florida
molecular weight
 molecular formula
 molecular structure ?

34. The large diversity of organic molecules
C16H10N2O2
MW =
Search molecular formula (= exact mass)
227 known isomers

35. etc ...
error ±0.02 Da: 8831 hits

36. Molecular structure in MS
Collision induced dissociation (MS/MS) *
collisional activation *
m/z
m/z
Fragment spectrum platform dependent
Fragment spectrum not predictable from first principles
MS/MS database comparison
Identify only known unknowns

37. Structural information from orthogonal methods
retention time (LCMS)
geometric cross section (ion mobility)
# labile H-atoms (H/D exchange)
IR spectroscopy
 connectivity
 intramolecular interactions
 predictable by QC
 database independent
IRIS MS

38. Complex sample (urine), isolate target compounds
With:
Radboud UMC
R. Wevers
L. Kluijtmans

39. Measure IR spectrum for biomarker ion isolated from sample
Measure IR spectra of biomarkers in complex mixture and compare to reference spectra
reference compound
Measure IR spectrum for biomarker ion isolated from sample

40. Can record IR spectra with sensitivity and selectivity of MS

41. IR spectrum of MS/MS product
-44 m/z

42. Distinguishing isomers
C=O stretch indicates that COOH group is not deprotonated

43. Conclusions IRMPD spectroscopy in Amazon ion trap
Good comparison to previously recorded spectra (FT-ICR)
Shorter acquisition times
Higher sensitivity
IR spectra of ions from ESI, CID, ETD
IR spectra of compounds isolated from complex mixture samples
Analytical applications

44. Acknowledgments FELIX staff Britta Redlich Lex van der Meer
Jonathan Martens
Lisanne Kempkes
Giel Berden
Josipa Grzetic
Christoph Gebhart (Bruker)
Ron Wevers (RUMC)
Leo Kluijtmans (RUMC)
NWO VICI
NWO Rekentijd