Bottom-Up Structural Analysis for Natural Products by Identifying Fragment Ions Resulted from Gas Phase Collision-Induced C–C Bond Fission

Structural degradation is a fundamental principle for identifying complicated structures. Except for the comparable ability with chemical degradation reactions to facilitate C-X (X = N, O, and S) bond fission, gas phase ion dissociation also enables C-C fission, leading to a bottom-up strategy to annotate complicated natural products via interpreting fragment ion structures. Here, fragmentation trajectories of the concerned first-generation fragment ions were universally recorded by operating online energy-resolved (ER)-MS in the second collision chamber of a QTRAP-MS device. Following the appropriate normalization, the breakdown graphs of the primary MS³ spectral signals composed of a full exciting energy ramp (FEER)-MS³ spectrum containing <i>m</i>/<i>z</i>, optimal exciting energy (OEE), EE at 50% survival yield (EE₅₀), and the maximal relative ion intensity (RII_OEE) features. Through involving diverse C-C fission routes from 77 compounds, three means were feasible to interpret fragment ions by FEER-MS³, such as 1) matching with FEER-MS³ of the known fragment ions from the authentic structures; 2) matching with FEER-MS² of the appropriate (de)protonated molecule that intactly crossed the front collision cell; and 3) deciphering <i>m</i>/<i>z</i>, OEE, EE₅₀, and RII_OEE to molecular descriptors using quantum structure calculation. Subsequently, the incorporation of a full collision energy ramp (FCER)-MS² spectrum that was built by programming ER-MS in the first collision cell, incorporating with the empirical mass fragmentation rules, enabled the entire structural configuration through linking substructures. The strategic utility was justified by Diels-Alder adduct-focused identification in <i>Morus alba</i>, and confidence-enhanced identification was reached for 63 compounds. Overall, FEER-MS³ significantly facilitated the identification of fragment ions generated by C-C bond fissions and advanced MS/MS-based structural annotation.