Christopher Leavitt Yale University Vibrational spectra of cryogenic peptide ions using H 2 predissociation spectroscopy

Motivation Characterize the effects of protonation in peptide ions Investigate the dependence of varying substituents across the peptide backbone to peptide conformation

Structural Probe: Methylation + H2OH2O H+H+ R= H, CH 3

Cryogenic Mass Spectrometry: H 2 -Tagging in a Quadrupole Ion Trap Wiley-McLaren extraction region Ion optics To time-of-flight and 2-D infrared analysis Electrospray needle Heated capillary 90°ion bender RF onlyquadrupolesH 2 /He filled 3-Dquadrupole ion trap with temperature control to 10 K Einzel Octopoles 1 st skimmer 2 nd skimmer Differential aperture 50 K heat shield 1x x x10 -7 Pressure (Torr) GlyGlyH + T = 300K T = 10K Ion Intensity (A.U.) Mass (m/z) * * * * * * * *

From ESI cm -1 2m Flight Tube MCP Detector Mass Gate Reflectron Yale Photofragmentation TOF Spectrometer Ion Optics A + · (H 2 ) m + h → A + · (H 2 ) n + (m-n) H 2

D0D0 Infrared Spectrum of GlyGlyH Photon Energy, cm -1 Predissociation Yield H 2 stretch Polfer, N. C., Oomens, J. Mass Spectrom. Rev. 2009, 28, Wu, R., McMahon, T. B. J. Phys. Chem. B 2009, 113, Kamrath, M., et. al. J. Am. Chem. Soc. 2011, 133, IVR IRMPD Room Temperature Tens to hundreds of photons are necessary to dissociation molecules

Infrared Spectrum of GlyGlyH Photon Energy, cm -1 Predissociation Yield H 2 stretch Wu, R., McMahon, T. B. J. Phys. Chem. B 2009, 113, Kamrath, M., et. al. J. Am. Chem. Soc. 2011, 133, IVR Cryogenic H 2 Predissociation Ions are vibrationally cold Single photon results in dissociation H2H2 H2H2

MP2/ G(d,p) Photon Energy, cm -1 Calculated Intensity Predissociation Yield H 2 stretch O-H stretch Protonated Amine N-H Region Amide Region Fingerprint Region Wu, R., McMahon, T. B. J. Phys. Chem. B 2009, 113, Kamrath, M., et. al. J. Am. Chem. Soc. 2011, 133, Infrared Spectrum of GlyGlyH +

n = 1 Predissociation Yield Photon Energy, cm -1 n = 2 Calculated Intensity n = 0 O-H stretch H 2 solvation of GlyGlyH + Asym. NH 2 stretch Amide NH Sym. NH 2 stretch O-H stretch Asym. NH 2 stretch Amide NH stretch Sym. NH 2 stretch Optimization and Frequency Calculations at MP2/6-311+G(d,p)

Structural Probe: Methylation ° GlyGlyH + (1) ° GlySarH + (1) ° SarSarH + (1) SarGlyH + (1) ° a)b) c)d) Optimization and Frequency Calculations at MP2/6-311+G(d,p) Extended, “all trans” Kinked, carboxyl rotated

Photon Energy, cm -1 Predissociation Yield O-H stretch Asym. NH 2 Amide NH Sym. NH 2 Amine NH GlyGlyH + (H 2 ) 1 SarSarH + (D 2 ) 2 SarGlyH + (H 2 ) 2 GlySarH + (D 2 ) 2 * * N-H Stretching Region: Methylation Study

Fingerprint Region: Methylation Study Photon Energy, cm -1 Predissociation Yield CO-H Bend Amide II C=O Amide I * * Optimization and Frequency Calculations at MP2/6-311+G(d,p) CO-H Bend Amide II Amide IC=O

Missing Shared Proton Bands MP2/ G(d,p) Photon Energy, cm -1 Calculated Intensity Predissociation Yield H 2 stretch O-H stretch Protonated Amine N-H Region Amide Region Fingerprint Region Wu, R., McMahon, T. B. J. Phys. Chem. B 2009, 113, Kamrath, M., et. al. J. Am. Chem. Soc. 2011, 133,

N-H Distance, Å Energy, cm -1 GlyGlyH + GlySarH + SarGlyH + SarSarH + H 2 N C H 2 C O H Identifying the Shared Proton Mode 00 1 cm -1 Optimization calculations at MP2/ G(d,p)

Identifying the Shared Proton Mode Predissociation Yield Photon Energy, cm -1

Photon Energy, cm -1 Identifying the Shared Proton Mode All structures are nominally protonated on the amino group, and feature an intramolecular H- bond between the amino group and the amide oxygen. Addition of a methyl group at the amide position induces rotation of the peptide backbone. Isotope substitution to help confirm the assignment of the intramolecular h-bond Isomer selective IR-IR double resonance experiments to determine the extent of multiple isomers present.

Thanks to: Mark Johnson Mike Kamrath Arron Wolk Etienne Garand Peter Jordan Rachael Relph Helen Gerardi Krissy Breen Andrew DeBlase Joe Fournier Gary Weddle Tim Guasco (UCSD) Mike Van Stipdonk (Wichita State) Anne McCoy (The Ohio State University) Acknowledgements

h probe Reflectron Signal Time of Flight, ms probe fragment pump fragment Detector Predissociation Dip Spectroscopy h pump (scanned) Coaxial TOF ±1.5 keV (fixed) Our Challenge: Not enough temporal separation! The Solution: Earlier first laser crossing and mass selection!