Photochemical and Collision-Induced Cross-Linking of Lys, Arg, and His to Nitrile Imines in Peptide Conjugate Ions in the Gas Phase

We report a study of internal covalent cross-linking with photolytically generated diarylnitrile imines of N-terminal arginine, lysine, and histidine residues in peptide conjugates. Conjugates in which a 4-(2-phenyltetrazol-5-yl)benzoyl group was attached to C-terminal lysine, that we call RAAA-<i>tet</i>-K, KAAA-<i>tet</i>-K, and HAAA-<i>tet</i>-K, were ionized by electrospray and subjected to UV photodissociation (UVPD) at 213 nm. UVPD triggered loss of N₂ and proceeded by covalent cross-linking to nitrile imine intermediates that involved the side chains of N-terminal arginine, lysine, and histidine, as well as the peptide amide groups. Cross-linking yields were determined from UVPD-MS² measurements as 67%, 66%, and 84% for RAAA-<i>tet</i>-K, KAAA-<i>tet</i>-K, and HAAA-<i>tet</i>-K ions, respectively. CID-MS³ of the denitrogenated ion intermediates from RAAA-<i>tet</i>-K, KAAA-<i>tet</i>-K, and HAAA-<i>tet</i>-K indicated overall cross-linking yields of 80%, 89%, and 80%, respectively. The nature of the cross-linking reactions and cross-link structures were investigated for RAAA-<i>tet</i>-K by high-resolution cyclic ion mobility mass spectrometry that identified precursor ion conformers and multiple dissociation products. All sequences were subjected to conformational analysis by Born-Oppenheimer molecular dynamics, and energy analysis by density functional theory calculations with M06-2X/def2qzvpp that provided relative and dissociation energies for several cross-link structural types. The cross-linking reactions were substantially exothermic, driving the efficient conversion of nitrile-imine intermediates to cyclic products. The principal steps in covalent cross-linking involved proton transfer onto the nitrile imine group accompanied by nucleophilic attack by the peptide side-chain and amide groups. Blocking the proton transfer and nucleophile resulted in a loss of cross-linking abilities.