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

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 cycliclization, modification, and fragmentation techniques.