Facile synthesis of Ti4+-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 4+ -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.