Photochemical and\nCollision-Induced Cross-Linking\nof Asp, Glu, Asn, and Gln Residues in Peptide-Nitrile Imine Conjugate\nIons in the Gas Phase

Peptide conjugates furnished with a 2,5-diaryltetrazolecarbonyl\ntag at the C-terminal lysine, which we call peptide-<i>tet</i>-K, were found to undergo efficient cross-linking of Asp, Glu, Asn,\nand Gln residues to transient nitrile-imine intermediates produced\nby photodissociation and collision-induced dissociation (CID) of the\ntetrazole ring in gas-phase ions. UV photodissociation (UVPD) at 213\nnm achieved cross-linking conversion yields of 37 and 61% for DAAAK-<i>tet</i>-K and EAAAK-<i>tet</i>-K, respectively. The\nyields for NAAAK-<i>tet</i>-K and QAAAK-<i>tet</i>-K were 29 and 57%, respectively. Even higher cross-link yields were\nfound for CID-MS<sup>3</sup> of stable denitrogenated ions, (peptide-<i>tet</i>-K–N<sub>2</sub> + H)<sup>+</sup>, that were in\nthe 69–83% range. Different types of cross-links were distinguished\nby CID-MS<sup>n</sup> that showed a distinct series of backbone fragment\nions, loss of N-terminal groups, and loss of phenylhydrazine from\nthe modified nitrile imines. The Asp and Glu side-chain carboxyl groups\nwere major participants in cross-linking that resulted in proton and\noxygen transfer to the nitrile imine group. Other types of cross-linking\ninvolved Asn and Gln <i>C</i>ONH<sub>2</sub> groups and\nbackbone amides. Cyclic ion mobility-mass spectrometry was used to\nseparate NAAAK-<i>tet</i>-K and QAAAK-<i>tet</i>-K conformers and products of their collision-induced denitrogenation.\nLinear nitrile-imine and cross-linked ion structures were identified\nby comparing the experimental collision cross sections (CC<i>S</i><sub>exp</sub>) to those for structures obtained by combined\nBorn–Oppenheimer molecular dynamics and density functional\ntheory (DFT) calculations. The formation of cross-links was found\nto be energetically favorable and involved proton-facilitated nucleophilic\nattack at the nitrile-imine carbon atom.