Photochemical and Collision-Induced Cross-Linking of Asp, Glu, Asn, and Gln Residues in Peptide-Nitrile Imine Conjugate Ions in the Gas Phase

Peptide conjugates furnished with a 2,5-diaryltetrazolecarbonyl tag at the C-terminal lysine, which we call peptide-<i>tet</i>-K, were found to undergo efficient cross-linking of Asp, Glu, Asn, and Gln residues to transient nitrile-imine intermediates produced by photodissociation and collision-induced dissociation (CID) of the tetrazole ring in gas-phase ions. UV photodissociation (UVPD) at 213 nm achieved cross-linking conversion yields of 37 and 61% for DAAAK-<i>tet</i>-K and EAAAK-<i>tet</i>-K, respectively. The yields for NAAAK-<i>tet</i>-K and QAAAK-<i>tet</i>-K were 29 and 57%, respectively. Even higher cross-link yields were found for CID-MS³ of stable denitrogenated ions, (peptide-<i>tet</i>-K-N₂ + H)⁺, that were in the 69-83% range. Different types of cross-links were distinguished by CID-MSⁿ that showed a distinct series of backbone fragment ions, loss of N-terminal groups, and loss of phenylhydrazine from the modified nitrile imines. The Asp and Glu side-chain carboxyl groups were major participants in cross-linking that resulted in proton and oxygen transfer to the nitrile imine group. Other types of cross-linking involved Asn and Gln <i>C</i>ONH₂ groups and backbone amides. Cyclic ion mobility-mass spectrometry was used to separate NAAAK-<i>tet</i>-K and QAAAK-<i>tet</i>-K conformers and products of their collision-induced denitrogenation. Linear nitrile-imine and cross-linked ion structures were identified by comparing the experimental collision cross sections (CC<i>S</i>_exp) to those for structures obtained by combined Born-Oppenheimer molecular dynamics and density functional theory (DFT) calculations. The formation of cross-links was found to be energetically favorable and involved proton-facilitated nucleophilic attack at the nitrile-imine carbon atom.