Scaffolds consisting of a peptide, a phthalate linker, and a 4,4-azipentyl group were synthesized and used to study intramolecular peptide–carbene cross-linking in gas-phase cations. Carbene intermediates were generated by UV-laser photodissociation at 355 nm of the diazirine ring in mass-selected ions, and the cross-linked products were detected and quantified by collision-induced dissociation tandem mass spectrometry (CID-MS n , n = 3–5). Peptide scaffolds containing Ala and Leu residues with a C-terminal Gly gave 21–26% yields of cross-linked products, while the presence of the Pro and His residues decreased the yields. Experiments using hydrogen–deuterium–hydrogen exchange, carboxyl group blocking, and analysis of CID-MS n spectra of reference synthetic products revealed that a significant fraction of cross-links involved the Gly amide and carboxyl groups. Interpretation of the cross-linking results was aided by Born–Oppenheimer molecular dynamics (BOMD) and density functional theory calculations that allowed us to establish the protonation sites and conformations of the precursor ions. Analysis of long (100 ps) BOMD trajectories was used to count close contacts between the incipient carbene and peptide atoms, and the counting statistics was correlated with the results of gas-phase cross-linking.