Efficient Isolation and Enrichment of SH2 Domain Protein Species from Plasma Exploiting Specific Peptide-Protein...

Fiber SiO₂ microspheres are modified with a phosphorylated peptide chain through a Schiff base reaction, and the product, pPeps@SiO₂ microspheres, is obtained. The interaction between the phosphorylated peptide chain and the specific site of the SH2 domain offers the as-prepared microspheres with good capture performance toward SH2 domain proteins. Under the condition of pH 4, the capture efficiencies of pPeps@SiO₂ microspheres for SH2-containing proteins SH2-SH2, SH2-SH3, and SH2-PTP are 91%, 61.3%, and 62.96%, respectively, which are much higher than those for proteins without an SH2 domain. The adsorbed SH2 proteins can be readily recovered by using 0.1 mol L^-1 imidazole; therefore, a strategy for the isolation of SH2 domain proteins from complex matrices is proposed. SDS-PAGE results indicate that the isolation of the SH2-SH3 protein from plasma using pPeps@SiO₂ microspheres is successfully achieved, and the isolation process is not affected by other high-abundance protein species in plasma. The concentration of SH2 domain protein in plasma increases from 12.4 pg mL^-1 to 61.59 pg mL^-1 after treating with pPeps@SiO₂ microspheres, which well demonstrates the favorable separation and enrichment ability of pPeps@SiO₂ microspheres. The isolation strategy based on protein domains not only provides the basis for in-depth investigation of SH2 domain protein functions but also provides new ideas for protein separation and purification.