Christopher LeavittYale University

Vibrational spectra of cryogenic peptide ions using H2 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

+

H2O

N

H

O

NHH

O

OH

O

O

NHH

HN

O

OHH

H

N

H

O

N+HH

O

OH

H

H+

N

CH3

O

N+HH

O

OH

H

R= H, CH3

N

R

O

N+RH

O

OH

H

N

H

O

N+HH

O

OH

H

N

H

O

N+H3C

H

O

OH

H

N

CH3

O

N+H3C

H

O

OH

H

Cryogenic Mass Spectrometry: H2-Tagging in a Quadrupole Ion Trap

Wiley-McLarenextraction region

Ion optics

To time-of-flightand 2-D infrared analysis

Electrosprayneedle

Heated capillary

90° ionbender

RF only quadrupolesH2/He filled 3-D quadrupoleion trap with temperature

control to 10 KEinzel

Octopoles

1st skimmer

2nd skimmerDifferential

aperture

50 K heat shield

1x10-5 1.5x10-23x10-7Pressure (Torr) 1.5 760

GlyGlyH+

T = 300K

T = 10K

Ion

Inte

nsity

(A

.U.)

Mass (m/z)

+H3N

O

HN

O

OH

1 2 3 4 5 6

* *

*

* * * * *133 137 141 145

FromESI

600-4500 cm-1

2m Flight TubeMCP Detector

Mass Gate

Reflectron

Yale Photofragmentation TOF Spectrometer

Ion Optics

A+ · (H2)m + h → A+ · (H2)n + (m-n) H2

D0

Infrared Spectrum of GlyGlyH+

800 1200 1600 2800 3200 3600 4000

Photon Energy, cm-1

Pre

dis

so

cia

tio

n Y

ield

H2 stretch

Polfer, N. C., Oomens, J. Mass Spectrom. Rev. 2009, 28, 468-494Wu, R., McMahon, T. B. J. Phys. Chem. B 2009, 113, 8767-8775Kamrath, 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

Infrared Spectrum of GlyGlyH+

800 1200 1600 2800 3200 3600 4000

Photon Energy, cm-1

Pre

dis

so

cia

tio

n Y

ield

H2 stretch

Wu, R., McMahon, T. B. J. Phys. Chem. B 2009, 113, 8767-8775Kamrath, M., et. al. J. Am. Chem. Soc. 2011, 133, 6440-6448

IVR

Cryogenic H2 Predissociation•Ions are vibrationally cold•Single photon results in dissociation

H2

H2

MP2/6-311++G(d,p)

800 1200 1600 2800 3200 3600 4000

Photon Energy, cm-1

Ca

lcu

late

d I

nte

ns

ity

Pre

dis

so

cia

tio

n Y

ield

H2 stretch

O-H stretch

Protonated Amine

freeH2

N-H Region

Amide Region

Fingerprint Region

Wu, R., McMahon, T. B. J. Phys. Chem. B 2009, 113, 8767-8775Kamrath, M., et. al. J. Am. Chem. Soc. 2011, 133, 6440-6448

Infrared Spectrum of GlyGlyH+

n = 1

Pre

dis

soci

atio

n Y

ield

3100 3200 3300 3400 3500 3600 3700

Photon Energy, cm-1

n = 2

Cal

cu

late

d I

nte

nsi

ty

n = 0

O-H stretch

H2 solvation of GlyGlyH+

Asym. NH2

stretch

Amide NH

Sym. NH2

stretch

O-Hstretch

Asym. NH2

stretch

Amide NH stretch

Sym. NH2

stretch

Optimization and Frequency Calculations at MP2/6-311+G(d,p)

Structural Probe: Methylation

1.065

1.689

127.2°

GlyGlyH+(1)

1.069

128.0°

1.654

GlySarH+(1)

1.8401.043

118.0°

SarSarH+(1)SarGlyH+(1)

1.9551.038

114.0°

a) b)

c) d)

Optimization and Frequency Calculations at MP2/6-311+G(d,p)

Extended,“all trans” Kinked,

carboxyl rotated

2400 2600 2800 3000 3200 3400 3600 3800 4000 4200

Photon Energy, cm-1

freeH2

freeD2

Pre

diss

ocia

tion

Yie

ld

O-H stretch

Asym. NH2

Amide NH

Sym. NH2

Amine NH

GlyGlyH+•(H2)1

SarSarH+•(D2)2

SarGlyH+•(H2)2

GlySarH+•(D2)2

*

*

N-H Stretching Region: Methylation Study

N

CH3

O

N+HH

O

OH

H

N

H

O

N+HH

O

OH

H

N

H

O

N+H3C

H

O

OH

H

N

CH3

O

N+H3C

H

O

OH

H

Fingerprint Region: Methylation Study

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900

Photon Energy, cm-1

Pre

diss

ocia

tion

Yie

ld

CO-HBend

Amide II C=O

Amide I

*

*

Optimization and Frequency Calculations at MP2/6-311+G(d,p)

CO-H Bend

Amide II

Amide I

C=O

N

CH3

O

N+HH

O

OH

H

N

H

O

N+HH

O

OH

H

N

H

O

N+H3C

H

O

OH

H

N

CH3

O

N+H3C

H

O

OH

H

Missing Shared Proton Bands

MP2/6-311++G(d,p)

800 1200 1600 2800 3200 3600 4000

Photon Energy, cm-1

Ca

lcu

late

d I

nte

ns

ity

Pre

dis

so

cia

tio

n Y

ield

H2 stretch

O-H stretch

Protonated Amine

freeH2

N-H Region

Amide Region

Fingerprint Region

Wu, R., McMahon, T. B. J. Phys. Chem. B 2009, 113, 8767-8775Kamrath, M., et. al. J. Am. Chem. Soc. 2011, 133, 6440-6448

0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

0

2500

5000

7500

10000

N-H Distance, Å

En

erg

y, c

m-1

GlyGlyH+

GlySarH+

SarGlyH+

SarSarH+

H2N

CH2

C

OH

Identifying the Shared Proton Mode

0

1

1555 cm-1

Optimization calculations at MP2/6-311++G(d,p)

Identifying the Shared Proton Mode

800 1000 1200 1400 1600 1800 2000

Pre

diss

ocia

tion

Yie

ld

Photon Energy, cm-1

N

H

O

NHH

O

OH

Na+

N

H

O

N+HH

O

OH

H

800 1200 1600 2400 2800 3200 3600

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 JohnsonMike KamrathArron WolkEtienne GarandPeter JordanRachael RelphHelen GerardiKrissy BreenAndrew DeBlaseJoe FournierGary WeddleTim Guasco(UCSD)Mike Van Stipdonk (Wichita State)Anne McCoy(The Ohio State University)

Acknowledgements

hprobe

Reflectron

Sig

nal

Time of Flight, ms

prob

e fr

agm

ent

pum

p fr

agm

ent

Detector

Predissociation Dip Spectroscopy

hpump

(scanned)

Coaxial TOF

±1.5 keV

(fixed)

Our Challenge:

Not enoughtemporal separation!

The Solution:

Earlier firstlaser crossingand mass selection!