An Integrated Strategy for Rapid Profiling of Depsipeptides by High-Performance Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry: <i>Bombyx batryticatus</i> as an Example

Depsipeptides, a structurally diverse class of nonribosomal peptides with broad bioactivities, present significant challenges for systematic characterization due to their complex fragmentation patterns in mass spectrometry (MS). This study aims to develop a generic three-step strategy based on high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS) to facilitate the rapid screening and identification of both linear and cyclic depsipeptides. The workflow is defined by three sequential, logic-driven steps: (1) filtering potential depsipeptide precursors via diagnostic and adduct ions along with depsipeptide classification; (2) identifying the structural residues by monitoring diagnostic product ions and neutral losses; and (3) sequencing the peptide backbone through comprehensive analysis of MS/MS spectra. To validate the strategy's universality, it was applied to the analysis of depsipeptides in a complex biological matrix (extracts of <i>Bombyx batryticatus</i>). A total of 62 depsipeptides (encompassing octa-, hexa-, tetra-, and didepsipeptides, with both cyclic and linear topologies) were identified or tentatively characterized, including 34 potential novel analogs. Notably, the strategy successfully distinguished 10 methionine-containing depsipeptides with subtle redox modifications (native methionine, methionine sulfoxide, methionine sulfone), demonstrating its ability to resolve structurally similar derivatives. This three-step strategy offers a simple, rapid, and robust tool for comprehensive depsipeptide profiling. Its application to complex matrices highlights the strong potential for extending to other biological samples, advancing systematic analysis of depsipeptide families in natural products and biological systems.