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

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

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.

UV photofragment spectroscopy of cryocooled ions B 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

[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, 11814.

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

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)

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

[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 0.945 in the hydride stretch, amide I, and amide II regions, respectively

[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

[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+]

[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

[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)

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.

[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

[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

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

Two low-energy conformers with same peptide backbone