Facile synthesis of Ti -immobilized affinity silica nanoparticles for the highly selective enrichment of intact phosphoproteins.

Currently, great challenges to top-down phosphoproteomics lie in the selective enrichment of intact phosphoproteins from complex biological samples. Herein, we developed a facile approach for synthesis of Ti -immobilized affinity silica nanoparticles and applied them to the selective separation and enrichment of intact phosphoproteins based upon the principle of metal(IV) phosphate/phosphonate chemistry. The as-prepared affinity materials exhibited high selectivity and adsorption capacities for model phosphoproteins (328.9 mg/g for β-casein, 280.5 mg/g for ovalbumin, and 225.8 mg/g for α-casein), compared with nonphosphoproteins (79.28 mg/g for horseradish peroxidase, 72.70 mg/g for BSA, and 27.28 mg/g for lysozyme). In addition, the resuability of the affinity silica nanoparticles was evaluated, and the results demonstrated a less than 10% loss of adsorption capacity after six adsorption-regeneration cycles. The practicability of the affinity materials was demonstrated by separating phosphoproteins from protein mixtures and drinking milk samples, and the satisfactory results indicated its potential in phosphoproteomics analysis.