Application of feature-based molecular networking and MassQL for the MS/MS fragmentation study of depsipeptides

Abstract

The Feature-based Molecular Networking (FBMN) is a well-known approach for mapping and identifying structures and analogues. However, in the absence of prior knowledge about the molecular class, assessing specific fragments and clusters requires time-consuming manual validation. This study demonstrates that combining FBMN and Mass Spec Query Language (MassQL) is an effective strategy for accelerating the decoding mass fragmentation pathways and identifying molecules with comparable fragmentation patterns, such as beauvericin and its analogues. To accomplish this objective, a spectral similarity network was built from ESI-MS/MS experiments of Fusarium oxysporum at various collision energies (CIDs) and paired with a MassQL search query for conserved beauvericin ions. FBMN analysis revealed that sodiated and protonated ions clustered differently, with sodiated adducts needing more collision energy and exhibiting a distinct fragmentation pattern. Based on this distinction, two sets of particular fragments were discovered for the identification of these hexadepsipeptides: ([M + H]⁺) m/z 134, 244, 262, and 362 and ([M + Na]⁺) m/z 266, 284 and 384. By using these fragments, MassQL accurately found other analogues of the same molecular class and annotated beauvericins that were not classified by FBMN alone. Furthermore, FBMN analysis of sodiated beauvericins at 70 eV revealed subclasses with distinct amino acid residues, allowing distinction between beauvericins (beauvericin and beauvericin D) and two previously unknown structural isomers with an unusual methionine sulfoxide residue. In summary, our integrated method revealed correlations between adduct types and fragmentation patterns, facilitated the detection of beauvericin clusters, including known and novel analogues, and allowed for the differentiation between structural isomers.

Keywords: MS/MS fragmentation; MassQL; PCA; beauvericin; feature-based molecular networking.

FIGURE 1

Principal Component Analysis (PCA) of the MS/MS beauvericin data at 50 eV and 70 eV. Analysis was performed using tandem MS data at different collision energies to obtain principal components (PCs) that explained the total beauvericin variation (A) 2D-Score plots of the PCs for 50 (PC3 and PC6) and 70 eV (PC1 and PC3). (B) 1D-Loading plots of the PCs that displayed a strong correlation was observed with beauvericin. For 50 eV, PC3 and PC6 show a combined variance of 16.95%, whereas for 70eV, PC1 and PC3 have 79.34% of explained variance.

FIGURE 2

Fragmentation of beauvericin as a protonated ion and sodiated adduct at 25, 50, and 70 eV. The structure is shown both in its linear and cyclic form to facilitate visualization. Breaks in the peptide bond of each amino acid residue are shown in different colors and correspond to the b-fragments.

FIGURE 3

FBMN of beauvercin analogues at different collision energies (25, 50, and 70 eV). Each dataset was obtained from replicates of the same fungi extract and contains a similar number of MS/MS features. Nodes are colored according to the mass of the parent ions, in which yellowish colors represent lower masses (∼m/z 250) and purple-like nodes constitute higher masses (∼m/z 1,200).

FIGURE 4

Elucidation of structural isomers from the beauvericin molecular class. Two sodiated ions at m/z 806 and m/z 792 have been clustered separately and contain different fragmentation patterns. The group colored in green belongs to the known compounds, beauvericin (m/z 806.4,031) and beauvericin D (m/z 792.3874) and elute at later retention time (∼38 min). The other group, colored in blue, elutes at earlier retention times (∼30 min) and belongs the to the novel beauvericin analogues (1, m/z 806.3695) and (2, m/z 792.3536). These metabolites contain an unusual methionine sulfoxide residue and a diagnostic loss of 64 Da, corresponding to the neutral loss of methanesulfenic acid. Both groups display the diagnostic beauvericin ions at m/z 266, 284, 384.

FIGURE 5

Protonated beauvericin clusters from the FBMN at 25, 50, and 70 eV. Nodes colored in yellow are found in all CIDs whereas nodes colored in gray are specific for only one CID. Example of the most abundant ions found on the protonated cluster. MS/MS spectra of the ions m/z 766 and m/z 770 are sequentially shown in all three collision energies. The diagnostic fragments for this molecular class are colored in the spectra as m/z 134, 244, 262, and 362.

FIGURE 6

Sodiated beauvericin clusters from FBMN at 25, 50, and 70 eV. Nodes can be tracked by their color in all three collision energies, except for the nodes colored in gray which are specific for only one CID energy. Example of the most abundant ions found on the protonated cluster. MS/MS spectra of the ions m/z 822 and m/z 792 are sequentially shown in all three collision energies. The diagnostic fragments for this molecular class are colored in the spectra as m/z 266, 284, and 384.