Integrated LC-MS/MS and molecular dynamics approach reveals calcium-binding behavior of collagen-derived peptides...

Fish bone-derived calcium may offer an alternative to conventional supplements, but the contribution of specific peptides to calcium bioavailability has not been clearly defined. This study aimed to valorize an underutilized silver sillago bone by identifying novel calcium-binding peptides using de novo LC-MS/MS, molecular dynamic simulation, and Caco-2 monolayer transport assay. Bio‑calcium from silver sillago was prepared through enzymatic hydrolysis, defatting, bleaching, and grinding, then characterized by X-ray diffraction and FTIR spectroscopy. Caco-2 monolayer transport was used for the bioavailability assay. Peptides released after simulated gastrointestinal digestion were analyzed using LC-MS/MS, and calcium-binding potential was assessed through 100 ns molecular dynamics simulations. XRD confirmed the presence of crystalline hydroxyapatite with peaks at 2θ = 26°-32°, while FTIR identified characteristic Amide I-III and phosphate vibration bands. Among the 20 most abundant peptides, we report three novel peptides from silver sillago (SW4, SW16, and SW17) that demonstrated the strongest calcium-binding potential, with Ca²⁺-peptide distances <0.35 nm, ≥1.2 close Ca²⁺ ions, and ≥ 25 atomic contacts per frame. SW4 exhibited the shortest mean distance (0.29 nm), highest Ca²⁺ proximity (1.76 ions), and ∼ 28 contacts/frame. In vitro Caco-2 monolayer transport assay showed that bio‑calcium achieved a calcium absorption rate of 28.63 ± 3.88%, comparable to that of calcium carbonate (27.67 ± 3.81%). These results suggest that collagen-derived peptides in silver sillago bone may contribute to calcium retention and transport in this in vitro model, but their specific roles in cellular uptake and relevance in vivo remain to be established.