Gas-Phase Folding of a Prototypical Protonated Pentapeptide: Spectroscopic Evidence for Formation of a Charge-Stabilized ß-Hairpin.

Ultraviolet and infrared-ultraviolet (IR-UV) double-resonance photofragment spectroscopy has been carried out in a tandem mass spectrometer to determine the three-dimensional structure of cryogenically cooled protonated C-terminally methyl esterified leucine enkephalin [YGGFL-OMe+H](+). By comparing the experimental IR spectrum of the dominant conformer with the predictions of DFT M05-2X/6-31+G(d) calculations, a backbone structure was assigned that is analogous to that previously assigned by our group for the unmodified peptide [ Burke, N.L.; et al. Int. J. Mass Spectrom. 2015 , 378 , 196 ], despite the loss of a C-terminal OH binding site that was thought to play an important role in its stabilization. Both structures are characterized by a type II' ß-turn around Gly(3)-Phe(4) and a γ-turn around Gly(2), providing spectroscopic evidence for the formation of a ß-hairpin hydrogen bonding pattern. Rather than disrupting the peptide backbone structure, the protonated N-terminus serves to stabilize the ß-hairpin by positioning itself in a pocket above the turn where it can form H-bonds to the Gly(3) and C-terminus C═O groups. This ß-hairpin type structure has been previously proposed as the biologically active conformation of leucine enkephalin and its methyl ester in the nonpolar cell membrane environment [ Naito, A.; Nishimura, K. Curr. Top. Med. Chem. 2004 , 4 , 135 - 143 ].

Strategies for generating peptide radical cations via ion/ion reactions.

Several approaches for the generation of peptide radical cations using ion/ion reactions coupled with either collision induced dissociation (CID) or ultraviolet photo dissociation (UVPD) are described here. Ion/ion reactions are used to generate electrostatic or covalent complexes comprised of a peptide and a radical reagent. The radical site of the reagent can be generated multiple ways. Reagents containing a carbon-iodine (C-I) bond are subjected to UVPD with 266-nm photons, which selectively cleaves the C-I bond homolytically. Alternatively, reagents containing azo functionalities are collisionally activated to yield radical sites on either side of the azo group. Both of these methods generate an initial radical site on the reagent, which then abstracts a hydrogen from the peptide while the peptide and reagent are held together by either electrostatic interactions or a covalent linkage. These methods are demonstrated via ion/ion reactions between the model peptide RARARAA (doubly protonated) and various distonic anionic radical reagents. The radical site abstracts a hydrogen atom from the peptide, while the charge site abstracts a proton. The net result is the conversion of a doubly protonated peptide to a peptide radical cation. The peptide radical cations have been fragmented via CID and the resulting product ion mass spectra are compared to the control CID spectrum of the singly protonated, even-electron species. This work is then extended to bradykinin, a more broadly studied peptide, for comparison with other radical peptide generation methods. The work presented here provides novel methods for generating peptide radical cations in the gas phase through ion/ion reaction complexes that do not require modification of the peptide in solution or generation of non-covalent complexes in the electrospray process.

UV photofragmentation and IR spectroscopy of cold, G-type ß-O-4 and ß-ß dilignol-alkali metal complexes: structure and linkage-dependent photofragmentation.

Ultraviolet photofragmentation spectroscopy and infrared spectroscopy were performed on two prototypical guaiacyl (G)-type dilignols containing ß-O-4 and ß-ß linkages, complexed with either lithium or sodium cations. The complexes were generated by nanoelectrospray ionization, introduced into a multistage mass spectrometer, and subsequently cooled in a 22-pole cold ion trap to T ≈ 10 K. A combination of UV photofragment spectroscopy and IR-UV double resonance spectroscopy was used to characterize the preferred mode of binding of the alkali metal cations and the structural changes so induced. Based on a combination of spectral evidence provided by the UV and IR spectra, the Li(+) and Na(+) cations are deduced to preferably bind to both dilignols via their linkages, which constitute unique, oxygen-rich binding pockets for the cations. The UV spectra reflect this binding motif in their extensive Franck-Condon activity involving low-frequency puckering motions of the linkages in response to electronic excitation. In the pinoresinol•Li(+)/Na(+) complexes involving the ß-ß linkage, the spectra also showed an inherent spectral broadening. The photofragment mass spectra are unique for each dilignol•Li(+)/Na(+) complex, many of which are also complementary to those produced by collision-induced dissociation (CID), indicating the presence of unique excited state processes that direct the fragmentation. These results suggest the potential for site-selective fragmentation and for uncovering fragmentation pathways only accessed by resonant UV excitation of cold lignin ions.