DEVELOPMENT OF Ti4+-IMMOBILIZED NANOPOROUS MONOLITHIC POLYMER FOR SELECTIVE SEPARATION AND DETECTION OF PHOSPHOPEPTIDES

摘要

Phosphopeptides present in a large number of biological regulating mechanisms. It plays a significant role in gene regulation, eukaryotic signal transduction, and metabolic control in a cell. Abnormal phosphorylation can cause various diseases, including cancer. However, phosphoprotein abundances are very low, only 1-2%. In this work, the nanoporous monolith columns were developed for separation and detection of phosphopeptides in phosphoproteome analysis. The nanoporous monolith-based column is prepared inside a silicosteel tubing (100 x 1.02 mm i.d.) by insitu copolymerization reaction of glycidyl methacrylate (GMA) with ethylene dimethacrylate (EDMA). This monolith was further chemically modified by introducing aminomethyl phosphonic acid (AMPA) before immobilization of Ti4+ ion (Ti4+-immobilized). The monolithic column properties, such as morphology, elemental analysis, surface area analysis, permeability and pore distribution were characterized in detail. Such a Ti4+ - immobilized nanoporous monolith-based column with immobilization time 3 hours of TiCl4 without glutaraldehyde addition was further applied to separation and detection of phosphopeptides from digested proteins (β-casein and cytochrome-c), and tyrosine phosphorylated peptide samples. The result demonstrated that Ti4+-immobilized nanoporous monolith-based provide higher selectivity and efficiency for selective detection of phosphopeptides using liquid chromatography.