Ti (IV) attached-phosphonic acid functionalized capillary monolith as a stationary phase for in syringe type fast and robust enrichment of phosphopeptides.

In this study, poly (vinylphosphonic acid-co-ethylene dimethacrylate), poly (VPA-co-EDMA) capillary monolith was synthesized as a starting material for obtaining a stationary phase for microscale enrichment of phosphopeptides. The chelation of active phosphonate groups with Ti (IV) ions gave a macroporous monolithic column with a mean pore size of 5.4 μm. The phosphopeptides from different sources were enriched on Ti (IV) attached-poly (VPA-co-EDMA) monolith by using a syringe-pump. The monolithic capillary columns exhibited highly sensitive/selective enrichment performance with the phosphoprotein concentrations as low as 1.0 fmol/mL. Six different phosphopeptides were detected with high intensity by the treatment of β-casein digest with the concentration of 1.0 fmol/mL, using Ti (IV)@poly (VPA-co-EDMA) monolith. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts, in a complex mixture of digested proteins (molar ratio of β-casein to BSA, 1:1500) and three phosphopeptides were successfully detected. Four highly intense signals of phosphopeptides in human serum were also observed with high signal to noise ratio and a clear background after enrichment with Ti (IV)@poly (VPA-co-EDMA) monolith. It was concluded that capillary microextraction system enabled fast, efficient, and robust enrichment of phosphopeptides from microscale complex samples. The whole enrichment process was completed within 20 min which was shorter than the previously reported studies.