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

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

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

FELIX - Free Electron Laser Int’l user facility www.ru.nl/felix 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

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

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

Application of ion spectroscopy: understanding peptide fragmentation

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

Experimental: modified Bruker Amazon Speed ETD

Experimental: modified Bruker Amazon Speed ETD From FEL To photodiode

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

Comparison ion trap vs. FT-ICR Amazon Pro3H+

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)

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

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

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

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

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.

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

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

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

Following the reaction network: deamidation of AsnAlaH+

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

Mapping the entire reaction network Lisanne Kempkes

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

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

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

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

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

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

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

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

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 ?

The large diversity of organic molecules C16H10N2O2 MW = 262.074 Search molecular formula (= exact mass) 227 known isomers

etc ... error ±0.02 Da: 8831 hits

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

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

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

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

Can record IR spectra with sensitivity and selectivity of MS

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

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

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

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