Mass Spectrometry-Based Structural Characterization and Reaction Kinetics Profiling of Stapled Peptides through Enzymatic Linearization.

Peptide stapling stabilizes bioactive conformations and expands the chemical space of macrocyclic peptide therapeutics. However, competing stapling pathways often generate structurally heterogeneous products, complicating connectivity assignment and quantitative analysis of macrocyclization selectivity. Here, we develop a mass spectrometry (MS)-based analytical platform that enables structural elucidation of stapled peptides and mechanistic interrogation of their formation pathways. The method employs MS-grade proteases to achieve controlled enzymatic linearization of stapled macrocycles under mild conditions, converting rigid cyclic architectures into linear surrogates that are amenable to tandem MS sequencing while preserving staple-dependent connectivity information. When integrated with ion mobility spectrometry, the workflow enables separation and quantitative monitoring of regioisomeric stapled products, allowing direct assessment of site-selective macrocyclization kinetics. Extension of the approach through covalent labeling further demonstrates its ability to localize modification sites within constrained mono- and bicyclic stapled peptides. Together, this platform enables the systematic analysis of stapling connectivity, topology, reaction selectivity, and chemical modification patterns in rigid stapled peptide systems, offering a valuable analytical tool for the characterization and optimization of macrocyclic therapeutics.