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

2 Ion spectroscopy: photo-dissociation as spectroscopic toolMass spectrum IR spectrum

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

4 FELIX - Free Electron LaserInt’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 toolPolfer, Oomens, PCCP 2007, 9, 3804

6 Application of ion spectroscopy: understanding peptide fragmentationcollisional 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 ETD1.) 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 ETDFrom FEL To photodiode

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

12 Comparison ion trap vs. FT-ICRAmazon Pro3H+

13 Bruker Amazon: advantages over our FTMSHigh 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 AsnHeaton, Armentrout, JASMS 2009, 20, 852

16 Deamidation of protonated Gln and AsnKempkes, 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, GlnAlaN-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, AlaGlnNH3-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 networkLisanne Kempkes

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

26 ETD as alternative for CID in peptide sequencingCoon, 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-radicala-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-radicalLower in energy b-radical Martens, Grzetic, Berden, Oomens, Nature Commun. 2016, 7, 11754

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

33 Mass spectrometry as analytical toolmetabolites 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 moleculesC16H10N2O2 MW = Search molecular formula (= exact mass) 227 known isomers

35 etc ... error ±0.02 Da: 8831 hits

36 Molecular structure in MSCollision 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 methodsretention 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 compoundsWith: Radboud UMC R. Wevers L. Kluijtmans

39 Measure IR spectrum for biomarker ion isolated from sampleMeasure 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 isomersC=O stretch indicates that COOH group is not deprotonated

43 Conclusions IRMPD spectroscopy in Amazon ion trapGood 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 MeerJonathan Martens Lisanne Kempkes Giel Berden Josipa Grzetic Christoph Gebhart (Bruker) Ron Wevers (RUMC) Leo Kluijtmans (RUMC) NWO VICI NWO Rekentijd