Photochemical and\nCollision-Induced Cross-Linking\nin Stereochemically Distinct Scaffolds of Peptides and Nitrile Imines\nin Gas-Phase Ions

Intramolecular cross-linking\nbetween peptides and nitrile-imine\nintermediates was studied in stereochemically distinct conjugates\nin which the reacting components were mounted on <i>cis</i>-1,2-cyclohexane and <i>trans</i>-1,4-cyclohexane scaffolds\nthat we call 1,2-<i>s</i>-peptides and 1,4-<i>s</i>-peptides, respectively. The nitrile-imine intermediates were generated\nby N<sub>2</sub> loss from 2,5-diaryltetrazole tags upon UV-photodissociation\nat 213 and 250 nm or by collision-induced dissociation, and further\ninterrogated by CID and UVPD-MS<sup>3</sup>. Peptide fragment ion\nseries originating from linear structures and macrocyclic cross-links\nwere distinguished and used to quantify the cross-linking yields.\nThe yields in MS<sup>2</sup> varied between 27% for AAAG conjugates\nto 78% for GAAAK conjugates, depending on the peptide sequence. The\nCID-MS<sup>3</sup> yields were in a 57–97% range, depending\non the peptide sequence. Structures of 1,2-<i>s</i>-peptide\nand 1,4-<i>s</i>-peptide ions as well as several of their\nnitrile-imine intermediates and cross-links were investigated by high-resolution\ncyclic ion mobility in combination with Born–Oppenheimer molecular\ndynamics and density functional theory calculations. Matches between\nthe experimental and calculated collision cross sections and ion relative\nGibbs energies were used to assign peptide structures. Peptide conjugates\nC-terminated with Gly and Lys residues underwent cross-linking by\nthe carboxyl group, as established by MS<sup>3</sup> sequencing and\ncorroborated by carboxyl blocking experiments that lowered the cross-linking\nyields. Peptide conjugates C-terminated with Arg also cross-linked\nvia the side-chain guanidine group. A notable feature of the 1,4-<i>s</i>-peptide ions was the participation of low-energy twist-boat\ncyclohexane conformers that was enforced by strong hydrogen bonds\nbetween the peptide and nitrile imine.