Synthesis and characterization of silica-based stationary phases for phosphoprotein isolation via immobilized metal affinity chromatography

A silica-based immobilized metal affinity chromatography (IMAC) sorbent functionalized with phosphonate group was synthesized and characterized. The designed sorbent was suitable for the purification of large phosphorylated biomolecules due to its porosity. Monodisperse-mesoporous silica microspheres were synthesized according to a staged shape-templated hydrolysis and condensation protocol. Poly(methacrylic acid-co-ethylene glycol dimethacrylate) (poly(MAA-co-EGDMA) microspheres was used as a template for synthesizing the support material, silica microspheres. After enrichment of hydroxyl groups, the support was functionalized with covalently bound ligand 3- tri(hydroxysilyl)propyl methylphosphonate (THSPMP) containing phosphonate groups. Ti(IV) cations were immobilized on the support via phosphonate moieties. The developed sorbent was used for the isolation of phosphoproteins using the affinity between metal (IV) cations and phosphate groups. The equilibrium adsorption capacity and desorption yield of Ti(IV) immobilized phosphonate- functionalized silica microspheres (Ti(IV)@THSPMP@SiO2) were determined using a-casein and b-casein as phosphoproteins target proteins, and bovine serum albumin as a reference protein in a batch system. The highest equilibrium adsorption capacity for a-casein, b-casein, and BSA were 597 mg phosphoprotein/g sorbent, 194 mg phosphoprotein/g sorbent, and 116 mg protein/g sorbent, respectively. Phosphoproteins were isolated from milk and human serum with high desorption yields (quantitative for 50-fold diluted milk and human serum) and high purity (95% in first desorption and 85% in second desorption for 50-fold diluted milk and 90% with a-casein and 93% with b-casein for human serum experiments). Microcolumn permeability was calculated as 3.66 x 10-14 m2 for the microfluidic system. Ti(IV)@THSPMP@SiO2 microspheres were evaluated as a stationary phase for dynamic chromatography based on their appropriate column permeability, appropriate average diameter (5.3 μm), high pore volume (0.948 cm3/g) and high surface area (163 m2/g.)