Cryogenic Spectroscopy and Quantum Molecular Dynamics Determine the Structure of Cyclic Intermediates Involved in Peptide Sequence Scrambling

Collision-induced dissociation (CID) is a key technique used in mass spectrometry-based peptide sequencing. Collisionally activated peptides undergo statistical dissociation, forming a series of backbone fragment ions that reflect their amino acid (AA) sequence. Some of these fragments may experience a “head-to-tail” cyclization, which after proton migration, can lead to the cyclic structure opening in a different place than the initially formed bond. This process leads to AA sequence scrambling that may hinder sequencing of the initial peptide. Here we combine cryogenic ion spectroscopy and <i>ab initio</i> molecular dynamics simulations to isolate and characterize the precise structures of key intermediates in the scrambling process. The most stable peptide fragments show intriguing symmetric cyclic structures in which the proton is situated on a <i>C</i>₂ symmetry axis and forms exceptionally short H-bonds (1.20 Å) with two backbone oxygens. Other nonsymmetric cyclic structures also exist, one of which is protonated on the amide nitrogen, where ring opening is likely to occur