1. Cold, Gas-Phase UV and IR Spectroscopy
69th International Symposium on Molecular Spectroscopy
11/19/2018
Cold, Gas-Phase UV and IR Spectroscopy
of Protonated Leucine Enkephalin and its Analogues
Graduate students:
Evan Buchanan (NIST)
Jacob Dean (Toronto)
Nathan Kidwell (Penn)
Deepali Mehta-Hurt
Joe Korn
Di Zhang
Joe Gord
Nicole Burke
Patrick Walsh
Daniel Hewett
John Hopkins
Post-doc:
Dr. Ryoji Kusaka (RIKEN)
Undergraduates:
Polina Novotnaya (Univ. Chicago)
Rachel Clayton (P&G)
Alex Parobek (Yale)
Lauren Myers
Yiming Xi
Collaborators:
Christian Müller (Bochum)
Sam Gellman (UW-Madison)
Scott McLuckey (Purdue)
Ned Sibert (UW-Madison)
David Plusquellic (NIST)
Lyudmila Slipchenko (Purdue)
Brooks Pate (UVa)
Ramesh Jasti (Univ. Oregon)
Providing a snap-shot of current progress on a study of leu-enkephalin, a prototypical pentapeptide.
“Standing in” for Nicole Burke, who is at ASMS this week.
Funding
11/19/2018 1 1

2. Instrument design
Scott
McLuckey
22-pole trap:
Design from
Tom Rizzo,
Oleg Boyarkine
q2 is used to isolate the parent via a notched chirp broadband waveform
q3 is used to isolate the generated photofragments with a broadband waveform, the photofragments can then be analyzed via MSAE or can be dumped as a single ion pack
Ions are generated via nano-electrospray ionization (nESI)
q2 and q3 = linear ion traps
The cold trap = 22-pole ion trap held at ~5K

3. Model system: YGGFL a4 x1 b1 b2 b3 b4 y4 y3 y2 y1 Y G F L
Protonated leucine enkephalin (m/z=556 Da): model system in mass spectrometry1
b4 ion is the lowest energy fragmentation pathway
One previous 300K IRMPD study of the parent2
Other work: focus on structures of the CID fragments
1 Sztaray, J.; Memboeuf, A.; Drahas, L.; Vekey, K.; Mass Spectrom. Rev. 2011, 30, 298.
2 Chen, X.; Steill, J.D.; Oomens, J.; Polfer, N.C.; J Am Soc Mass Spectrom 2010, 21, 1313.

4. UV photofragment spectroscopy of cryocooled ionsB
Protonated tyrosine
(Frag1+H)++ X
Excited
State
Fragmentation
(M+H)+*
(M+H)+ S0 S1
Dissociation
threshold
Scan UV
laser
Unimolecular
Dissociation
(Frag2+H)+ +Y
Internal
Conversion
(M+H)+‡
Conformer B
Conformer A

5. [YGGFL+H]+ UV Photofragment Spectrum
Intensity (arb. unit)
3 cm-1
Three sharp transitions between 35,830 and 35,860 give average FWHM of 3 cm-1; the 10 cm-1 wide peak at 35,684 cm-1 shows splitting at higher resolution
Wavenumbers (cm-1)
Origin at 35,684 cm-1: ~600 cm-1 blue shifted from protonated tyrosine 3,4
Presence of more than one conformer with similar backbone
3 Redwine, J. G.; Davis, Z. A.; Burke, N. L.; Ogelsbee, R. A.; McLuckey, S. A.; Zwier, T. S.; Int J Mass Spectrom 2013, 348, 9.
4 Stearns, J. A.; Mercier, S.; Seaiby, C.; Guidi, M.; Boyarkin, O. V.; Rizzo, T. R.; J Am Chem Soc 2007, 129,

6. IR-UV Double Resonance: Single-conformation IR spectroscopy
Excited
State
Fragmentation
(Frag1+H)++ X S1
(M+H)+*
Dissociation
threshold
Internal
Conversion
(M+H)+‡
(Frag2+H)++Y
Unimolecular
Dissociation
Fix UV
dissociation
laser
on cold
transition
Scan IR
Laser
Dt=100 ns S0
(M+H)+
UV wavelength fixed
IR depletes cold
UV ion signal

7. IR-UV holeburn spectrum Comparison with heating the trap
a) 25 K
b) 50 K, signal 5x
Intensity (Arb. Units)
c) 110 K, signal 5x
d) IR excitation
3354 cm-1
Wavenumber (cm-1)

8. IR-induced Fragment Ion Gain Spectroscopy Contributions to spectrum from all conformers present
Excited
State
Fragmentation
(Frag1+H)++ X S1
(M+H)+*
Dissociation
threshold
Internal
Conversion
(M+H)+‡
(Frag2+H)++Y
Unimolecular
Dissociation
Fix UV
dissociation
laser to the
red of cold
transitions
IRFIGS or FIGIRS?
Scan IR
Laser
Dt=100 ns S0
(M+H)+
IR produces “warm”
ion gain signal
UV wavelength fixed

9. [YGGFL+H]+ IR FIG spectrum
NH3+ stretches
COO-H str
Ph-OH C7
“F”
C14
C10 p
C17/AS
C11/SS
Alkyl CH b)
Wavenumbers (cm-1)
Frag Ion Intensity (arb. units) a)
L C=O
Y C=O
G1 C=O
G2 C=O
δ(NH)
ring
F C=O
δ(NH3+)
MO5-2X/6-31*G(d) scaled 0.937, 0.955, and in the hydride stretch, amide I, and amide II regions, respectively

10. [YGGFL+H]+ Structure NH3+ ties up C=O groups, flip OH orientation
p_C17_C11 / C14 / C7 / F / C10 / C7(OH)
NH3+(a)_(b)_(c)/ NH(1)/NH(2)/NH(3)/NH(4)/COOH
The structure is 11.4 kJ/mol lower in energy and a better spectral match than the structure previously assigned from 300K IRMPD
NH3+ ties up C=O groups, flip OH orientation
7 H-bonds, some nearest neighbor, some remote
The NH3+ →O=C-OH→O=C(F) H-bonded chain forms a conduit that may be relevant to the b4 ion formation pathway

11. [YGGFL-OMe+H]+ UV Photofragment spectrum
Photofragment Ion Intensity
(arb. unit)
Wavenumbers (cm-1)
Origin at 35,600 cm-1 is red shifted 84 cm-1 from [YGGFL+H+]

12. [YGGFL-OMe+H]+ IR ion-gain spectrum
NH3+/C-H stretch
N-H stretch region
Ph-OH
Protonated YGGFL-OMe
NH3+
Intensity (arb. unit) F
C10
Protonated YGGFL
COO-H
NH3+ p C7
C14
Wavenumbers (cm-1)
Probable presence of two low energy conformers needs to be confirmed via IR depletion

13. [YGGFL-OMe+H]+ IR ion-gain spectrum
N-H stretch region
Ph-OH
NH3+/C-H stretch
A: 0.00 kJ/mol
B: 9.65 kJ/mol
Wavenumbers (cm-1) F C7
C10/C14
C10
C11
C14
C17 C7
Tentative assignment: Needs single-conformation spectra, mid-IR
NH3+ tying up backbone
6 H-bonds (no COOH)

14. UV Photofragmentation mass spectra
-107 b4 a4
y2/b3 y2
m/z b3
Intensity (Arb. Units)
[YGGFL+H]+
[YGGFL-OMe+H]+
Unique to
photofragment
Common w/ CID
Similar fragmentation patterns, intensities.
Still favors b4/a4 formation.

15. [YGGFL+Na]+ UV Photofragment spectrum
Intensity (arb. unit)
Neutral p-cresol
S0-S1 origin
Wavenumbers (cm-1)
Possible first origin at 35,290 cm-1: Red-shifted ~40 cm-1 relative to neutral
p-cresol Na+ not associated with tyrosine
Dense vibronic structure
More than one conformer probable

16. [YGGFL+Na]+ IR ion-gain spectrum
Sodiated YGGFL
Protonated YGGFL
N-H stretch region
Ph-OH
Free
COO-H
C-H stretch
H-bonded
COO-H, NH3+
Wavenumbers (cm-1)
Intensity (arb. unit)
More than one conformer probable, now with neutral NH2 + free COOH
Calculations in progress

17. The Zwier Group Acknowledgements Ned Sibert David Plusquellic Scott
Sam
Gellman
Scott
McLuckey

18. Two low-energy conformers with same peptide backbone