Application of High-Resolution Mass Spectrometry for the Metabolomic Profiling of Stress Responses in Marine Algae Under Heavy Metal Exposure

Marine microalgae serve as critical primary producers in aquatic ecosystems, yet their metabolic stability is increasingly threatened by anthropogenic heavy metal contamination. In this study, an untargeted metabolomics workflow based on ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS) was deployed to systematically elucidate the biochemical perturbations in the model diatom Phaeodactylum tricornutum exposed to sub-lethal concentrations of cadmium (Cd²⁺, 5.0 µM) and copper (Cu²⁺, 2.0 µM). Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) revealed pronounced, metal-specific metabolomic shifts. Over 120 statistically differential metabolites (variable importance in projection > 1.0, p < 0.05) were structurally annotated using accurate mass, isotopic pattern filtering, and MS/MS fragmentation matching. Cadmium exposure primarily disrupted sulfur assimilation and antioxidant machinery, evidenced by a dramatic depletion of reduced glutathione (GSH), significant accumulation of glutathione disulfide (GSSG), and the de novo induction of phytochelatin precursors (e.g., γ-glutamylcysteine). Conversely, copper stress initiated intense lipid peroxidation cascades, causing marked degradation of monogalactosyldiacylglycerols (MGDGs) and compensatory enrichment of sulfoquinovosyldiacylglycerols (SQDGs) alongside pronounced accumulations of proline and betaine osmolytes. These findings underscore the resolving power of HRMS for dissecting ecotoxicological mechanisms and establish specific molecular biosignatures for environmental biomonitoring of marine metal toxicity.