Highly selective titanium (IV)‐immobilized O‐phospho‐L‐tyrosine modified magnetic nanoparticles for the enrichment of intact phosphoproteins

Abstract

Phosphorylation is one of the most important protein post‐translational modifications, which possesses dramatic regulatory effects on the function of proteins. In consideration of the low abundance and low stoichiometry of phosphorylation and non‐specific signal suppression, efficient capture of the phosphoproteins from complex biological samples is critical to meet the need for protein profiling. In this work, a facile preparation of titanium (IV)‐immobilized O‐phospho‐L‐tyrosine modified magnetic nanoparticles was developed for the enrichment of intact phosphoproteins. The prepared magnetic nanoparticles were characterized by various instruments and had a spherical shape with an average diameter of 300 nm. The adsorption isotherms were investigated and the maximum capacity for β‐casein was calculated to be 961.5 mg/g. Standard protein mixtures and biological samples (non‐fat milk and human serum) were selected to test the enrichment performance. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis demonstrated the excellent enrichment performance with high selectivity. With the superparamagnetic property, titanium (IV)‐immobilized O‐phospho‐L‐tyrosine modified magnetic nanoparticles were convenient for the practical application and clinical promotion, thus having a promising prospect in the field of phosphoprotein research.

One‐step enrichment and stepwise elution of glycoproteins and phosphoproteins by hydrophilic Ti4+‐immobilized dendrimer poly(glycidyl methacrylate) microparticles functionalized with polyethylenimine and phytic acid

“…The phosphoprotein enrichment feasibility was studied in protein mixture of BSA and β-casein with the mass ratio of 1:1. The enrichment protocol was according to our previous report [26][27][28][29] with minor modifications and described in Supporting Information. The enrichment performance was tested by SDS-PAGE, which was conducted in Bio-Rad electrophoresis system (Bio-Rad Laboratories), and SDS-PAGE pattern was obtained by Amersham Imager 600 (GE Healthcare Life Sciences).…”