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

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

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

4. 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 1x10 -5 1.5x10 -2 3x10 -7 Pressure (Torr) 1.5760 GlyGlyH + T = 300K T = 10K Ion Intensity (A.U.) Mass (m/z) 123456 * * * * * * * * 133137141145

5. From ESI 600-4500 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

6. D0D0 Infrared Spectrum of GlyGlyH + 800120016002800320036004000 Photon Energy, cm -1 Predissociation Yield H 2 stretch Polfer, N. C., Oomens, J. Mass Spectrom. Rev. 2009, 28, 468-494 Wu, R., McMahon, T. B. J. Phys. Chem. B 2009, 113, 8767-8775 Kamrath, M., et. al. J. Am. Chem. Soc. 2011, 133, 6440-6448 IVR IRMPD Room Temperature Tens to hundreds of photons are necessary to dissociation molecules

7. Infrared Spectrum of GlyGlyH + 800120016002800320036004000 Photon Energy, cm -1 Predissociation Yield H 2 stretch Wu, R., McMahon, T. B. J. Phys. Chem. B 2009, 113, 8767-8775 Kamrath, M., et. al. J. Am. Chem. Soc. 2011, 133, 6440-6448 IVR Cryogenic H 2 Predissociation Ions are vibrationally cold Single photon results in dissociation H2H2 H2H2

8. MP2/6-311++G(d,p) 800120016002800320036004000 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, 8767-8775 Kamrath, M., et. al. J. Am. Chem. Soc. 2011, 133, 6440-6448 Infrared Spectrum of GlyGlyH +

9. n = 1 Predissociation Yield 3100320033003400350036003700 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)

10. Structural Probe: Methylation 1.065 1.689 127.2° GlyGlyH + (1) 1.069 128.0° 1.654 GlySarH + (1) 1.840 1.043 118.0° SarSarH + (1) SarGlyH + (1) 1.955 1.038 114.0° a)b) c)d) Optimization and Frequency Calculations at MP2/6-311+G(d,p) Extended, “all trans” Kinked, carboxyl rotated

11. 2400260028003000320034003600380040004200 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

12. Fingerprint Region: Methylation Study 8009001000110012001300140015001600170018001900 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

13. Missing Shared Proton Bands MP2/6-311++G(d,p) 800120016002800320036004000 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, 8767-8775 Kamrath, M., et. al. J. Am. Chem. Soc. 2011, 133, 6440-6448

14. 0.60.81.01.21.41.61.82.02.2 0 2500 5000 7500 10000 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1 1555 cm -1 Optimization calculations at MP2/6-311++G(d,p)

15. Identifying the Shared Proton Mode 800100012001400160018002000 Predissociation Yield Photon Energy, cm -1

16. 800120016002400280032003600 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.

17. 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

18. 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!