Guanidinium functionalized superparamagnetic silica spheres for selective enrichment of phosphopeptides and intact phosphoproteins from complex mixtures.

Phosphorylation of protein regulates nearly all biological processes in nature. The development of enrichment techniques for phosphorylated proteins is vital to systematic identification and characterization of phosphoproteins. Here, a general strategy for highly efficient capture of intact phosphorylated proteins from protein mixtures has been developed by using guanidine functionalized superparamagnetic microspheres (denoted as Fe3O4@SiO2@GDN). The Fe3O4@SiO2@GDN was prepared by modifying Fe3O4@SiO2 with 3-guanidopropyl triethoxysilane as a functionalization monomer. The resulting materials could specifically and selectively recognize phosphoproteins and showed high binding capacities for model phosphoproteins (78.8 mg g-1 for ovalbumin (OVA) and 59.6 mg g-1 for β-casein (β-Cas), respectively). The feasibility of the resulting material for phosphoprotein enrichment has also been demonstrated by selectively binding and capturing phosphoproteins from complex protein mixtures and real samples (milk, egg, and tissue protein extract from a mouse liver), respectively. In addition, the selective enrichment of phosphopeptides has also been investigated. The proposed technique showed application potential for phosphoprotein and phosphopeptide enrichment.