Cyclic Peptide Conformational Landscapes Through the Lens of Mass Spectrometry and Orthogonal Spectroscopy

ABSTRACT Cyclic peptides occupy a structurally dynamic region of chemical space, where function often arises from ensembles rather than a single dominant fold. Closed backbones, frequent N ‐methylation, heteroatoms, unnatural amino acids, and a strong propensity for metal coordination create dense conformational and coordination landscapes that frustrate crystallography and complicate ensemble analysis by solution NMR. This review charts the evolution of mass spectrometry (MS) into a powerful platform for mapping these landscapes across microsecond‐to‐millisecond timescales. We highlight high‐resolution ion mobility spectrometry (HR‐IMS) as a front‐end separator for non‐interconverting conformers, topoisomers, and coordination isomers, and show how mobility selection, combined with ion activation, disentangles kinetically trapped states from rapidly equilibrating ensembles. Because IMS alone cannot fully determine structure, we emphasize an integrated workflow that pairs IMS with complementary constraints: gas‐phase transition‐metal Förster resonance transfer for site‐specific distances, hydrogen–deuterium exchange (HDX), and solution NMR for residue‐level ensembles and exchange kinetics, and tandem MS and ion spectroscopy (IRMPD and cryogenic messenger tagging) for conformer‐specific vibrational fingerprints. Drawing on examples spanning rigid scaffolds such as gramicidin S and somatostatin, isotopomers of lasso peptides, conformationally flexible cyclosporines, the ionophoric depsipeptide beauvericin, and stapled peptides, we present an evidence ladder in which convergent gas‐ and solution‐phase observables enable confident structural assignments and discovery of new structural motifs with novel functions. We conclude with practical guidance on matching methods to questions and a forward‐looking perspective on time‐resolved and ensemble‐aware structural MS of macrocycles.