| Abstract
| - The gas-phase structures of the protonated pentapeptide Leu-enkephalin and its main collision-induced dissociation (CID) product ions, b4 and a4, are investigated by means of infrared multiple-photondissociation (IR-MPD) spectroscopy and detailed molecular mechanics and density functional theory (DFT)calculations. Our combined experimental and theoretical approach allows accurate structural probing ofthe site of protonation and the rearrangement reactions that have taken place in CID. It is shown that thesingly protonated Leu-enkephalin precursor is protonated on the N-terminus. The b4 fragment ion formstwo types of structures: linear isomers with a C-terminal oxazolone ring, as well as cyclic peptide structures.For the former structure, two sites of proton attachment are observed, on the N-terminus and on theoxazolone ring nitrogen, as shown in a previous communication (Polfer, N. C.; Oomens, J.; Suhai, S.;Paizs, B. J. Am. Chem. Soc. 2005, 127, 17154−17155). Upon leaving the ions for longer radiative coolingdelays in the ion cyclotron resonance (ICR) cell prior to IR spectroscopic investigation, one observes agradual decrease in the relative population of oxazolone-protonated b4 and a corresponding increase inN-terminal-protonated b4. This experimentally demonstrates that the mobile proton is transferred betweentwo sites in a gas-phase peptide ion and allows one to rationalize how the proton moves around the moleculein the dissociation process. The a4 fragment, which is predominantly formed via b4, is also confirmed toadopt two types of structures: linear imine-type structures, and cyclic structures; the former isomers areexclusively protonated on the N-terminus in sharp contrast to b4, where a mixture of protonation sites wasfound. The presence of cyclic b4 and a4 fragment ions is the first direct experimental proof that fully cyclicstructures are formed in CID. These results suggest that their presence is significant, thus lending strongsupport to the recently discovered peptide fragmentation pathways (Harrison, A. G.; Young, A. B.; Bleiholder,B.; Suhai, S.; Paizs, B. J. Am. Chem. Soc.2006, 128, 10364−10365) that result in scrambling of the aminoacid sequence upon CID.
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