IMSS: An Integrated Mass Spectrometry Strategy for Targeted Discovery of CyclopeptidesExemplified by Phakellicyclins from the Marine Sponge Phakellia fusca

Abstract Cyclopeptides (CPs) represent a structurally diverse class of bioactive natural products but remain analytically challenging due to their high structural similarity, low abundance, and complex matrices. Herein, we report an integrated mass spectrometry strategy (IMSS) enabling selective detection, annotation, and targeted isolation of CPs from marine organisms. The workflow incorporates: (1) multiplexed diagnostic neutral loss and precursor ion (NL/PI) scanning based on the six most frequent amino acid residues (Pro, Ile/Leu, Val, Tyr, Phe, Trp) identified from 16 reference standards; (2) de novo sequencing-assisted spectral validation via CycloNovo; (3) dual dereplication combining GNPS molecular networking with a curated library of 538 marine CPs; and (4) MS-triggered automated purification for target-oriented isolation. When applied to the marine sponge Phakellia fusca, IMSS afforded 23 candidate CPs and allowed targeted purification of two unknown cyclooctapeptides, phakellicyclins A and B, featuring rare dioxindolylalanine and β-3-oxindolylalanine residues, respectively. Both compounds exhibited potent anti-inflammatory activity through selective inhibition of interleukin-6 (IL-6) production without obvious cytotoxicity. This study establishes a robust and reproducible MS workflow for the targeted discovery of Pro/Ile/Leu-enriched marine CPs, with demonstrated potential for adaptation to other CP classes.