Microfluidic immobilized metal affinity chromatography based on Ti(IV)-decorated silica microspheres for purification of phosphoproteins

A silica-based immobilized metal affinity chromatography (IMAC) sorbent with the morphological properties suitable for purification of large phosphorylated biomolecules was synthesized. The sorbent was designed in the form of monodisperse-porous silica microspheres, 5.3 μm in size, having bimodal pore size distribution with a large median pore size (40 nm) and high surface area (163 m²/g) decorated with Ti(IV) cations (i.e. Ti(IV)@THSPMP@SiO₂ microspheres). The decoration of silica microspheres with Ti(IV) cations was made by using 3-(trihydroxysilyl)propyl methylphosphonate (THSPMP) as a bifunctiontional linker, by preserving their bimodal pore size distribution. The mesopores provided a large surface area for parking of adsorbed phosphoproteins as large phosphorylated biomolecules while the intraparticular transport of phosphoproteins was facilitated by the macropores providing a large median pore size. High equilibrium adsorption capacity and high desorption yield in the purification of phosphoproteins were obtained using Ti(IV)@THSPMP@SiO₂ microspheres as the sorbent in batch- and microfluidic-IMAC systems. The phosphoproteins, α-casein and β-casein were isolated from milk and human serum with almost quantitative yields and high purity in the batch IMAC system. The appropriate microcolumn permeability (3.66 × 10⁻¹⁴ m²) originating from its appropriate average diameter (5.3 μm), high porosity (0.948 cm³/g) and high surface area (163 m²/g) of Ti(IV)@THSPMP@SiO₂ microspheres makes the synthesized sorbent a promising stationary phase for dynamic chromatography. Hence, a new phosphoprotein enrichment format, a microfluidic IMAC system was constructed and successfully operated for highly selective purification of phosphoproteins from non-fat milk as a complex sample. The microfluidic-IMAC system is a promising tool particularly for phosphoproteomic applications performed using samples in microliter or nanoliter scale, also involving an on-line connection of purification unit to LC-MS for the identification of large phosphorylated biomolecules enriched.