Photochemical and\nCollision-Induced Cross-Linking\nof Lys, Arg, and His to Nitrile Imines in Peptide Conjugate Ions in\nthe Gas Phase

We report a study of internal covalent cross-linking\nwith photolytically\ngenerated diarylnitrile imines of N-terminal arginine, lysine, and\nhistidine residues in peptide conjugates. Conjugates in which a 4-(2-phenyltetrazol-5-yl)benzoyl\ngroup 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\n(UVPD) at 213 nm. UVPD triggered loss of N<sub>2</sub> and proceeded\nby covalent cross-linking to nitrile imine intermediates that involved\nthe side chains of N-terminal arginine, lysine, and histidine, as\nwell as the peptide amide groups. Cross-linking yields were determined\nfrom UVPD-MS<sup>2</sup> 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-MS<sup>3</sup> of the denitrogenated ion\nintermediates from RAAA-<i>tet</i>-K, KAAA-<i>tet</i>-K, and HAAA-<i>tet</i>-K indicated overall cross-linking\nyields of 80%, 89%, and 80%, respectively. The nature of the cross-linking\nreactions and cross-link structures were investigated for RAAA-<i>tet</i>-K by high-resolution cyclic ion mobility mass spectrometry\nthat identified precursor ion conformers and multiple dissociation\nproducts. All sequences were subjected to conformational analysis\nby Born–Oppenheimer molecular dynamics, and energy analysis\nby density functional theory calculations with M06-2X/def2qzvpp that\nprovided relative and dissociation energies for several cross-link\nstructural types. The cross-linking reactions were substantially exothermic,\ndriving the efficient conversion of nitrile-imine intermediates to\ncyclic products. The principal steps in covalent cross-linking involved\nproton transfer onto the nitrile imine group accompanied by nucleophilic\nattack by the peptide side-chain and amide groups. Blocking the proton\ntransfer and nucleophile resulted in a loss of cross-linking abilities.