Spatial comparison of the molecular composition of copper-binding organic ligands isolated by Cu(II)-IMAC-SPE and detected by Tims-TOF MS

The molecular composition of copper(II)-binding organic ligands isolated via immobilized metal affinity chromatography (IMAC) and solid-phase extraction (SPE-PPL) was investigated across distinct oceanic regions using trapped ion mobility spectrometry time-of-flight mass spectrometry (Tims-TOF). Significant differences in molecular composition were observed between high-nutrient, low-chlorophyll (HNLC; northeastern Pacific and southern Atlantic Oceans) and non-HNLC regions (Arctic Ocean). Non-HNLC samples exhibited higher mass-to-charge (m/z) values, elevated abundances of CAS-like and lignin-like compounds, and lower weighted CCS and H/C ratios, indicating greater aromaticity and a predominance of allochthonous organic ligands. The IMAC-SPE Cu(II)-binding ligand pools in the northeastern Pacific and southern Atlantic Oceans (HNLC regions) shared over 25% more compounds than those in the non-HNLC Arctic Ocean. Among the 17 compounds detected in all samples, carbazomycin A was identified with high confidence, marking its first detection in marine waters. Empirical CCS–m/z relationships revealed that IMAC-SPE Cu(II)-binding ligands exhibited strong, consistent correlations within the same molecular groups (carbohydrate-, CAS-, and lignin-like). The persistence of these relationships regardless of sample location and depth suggests that the ion mobility signatures are broadly conserved. Such consistency underscores the robustness of CCS–m/z trends, while also highlighting how molecular class exerts a stronger influence on IMAC-SPE Cu(II)-binding behavior than environmental origin. These findings underscore the importance of regional variability in IMAC-SPE Cu(II)-binding organic ligand composition and its implications for Cu bioavailability, toxicity, and global biogeochemical cycling in marine ecosystems.