Structure-aware fragment assignment for interpreting tandem mass spectrometry of modified and cyclic peptides

Abstract

Peptidomimetics such as fatty-acid-modified, head-to-tail cyclic, and disulfide-constrained peptides challenge conventional MS/MS interpretation because their fragmentation pathways extend beyond canonical backbone cleavages or require multiple bond breakages to generate sequence-informative ions. We present a structure-aware algorithm that calculates and labels theoretical fragment ions across modified and cyclic peptides, including side-chain and disulfide-related fragments. To evaluate assignments, we integrate the three numerical metrics sequence coverage, intensity coverage, and signal coverage and assess their behavior across m/z tolerances, intensity thresholds, and charge states. Using representative MS/MS data of angiotensin related peptides, liraglutide, semaglutide, cyclosporine, oxytocin, and somatostatin, we demonstrate that the metrics reliably distinguish correct from incorrect assignments, including closely related sequences. Incorporating fatty-acid-specific fragments increased intensity coverage for liraglutide and semaglutide. MS3 improved sequence coverage for the cyclic peptide cyclosporine relative to MS2, but no additional benefit was seen at MS4 level. For disulfide-bonded peptides, the combination of electron-transfer dissociation (ETD) and collision induced dissociation (CID) shifted fragment distributions toward disulfide-cleavage products relative to CID, which were captured by the algorithm’s dedicated disulfide labels. Together, these results demonstrate that structure-aware fragment calculation coupled to explicit assignment metrics enables more comprehensive and standardized interpretation of peptidomimetic MS/MS data. With this approach we lay a foundation for reproducible benchmarking which can be extended to additional cyclization, modification, and fragmentation techniques.