Enrichment of intact phosphoproteins using immobilized titanium(IV) affinity chromatography microspheres

Abstract

AbstractApproximately a third of eukaryotic proteins are estimated to be phosphorylated, and the protein phosphorylation is significant in regulating almost all aspects of cell life. Due to low stoichiometry of phosphorylation, efficient enrichment is often an essential step in phosphoproteomics study. Over the decades, lots of enrichment materials have been successfully developed and widely used for intact phosphoproeins. One of these materials is immobilized titanium(IV) affinity chromatography (Ti4+‐IMAC) microspheres, which has been widely studied and used for phosphopeptides enrichment. Application of Ti4+‐IMAC for intact phosphoprotein enrichment has also proven possible, where intact phosphoproteins (α‐casein and β‐casein) were successfully enrichment out of their mixture with BSA (1:1:10, w/w). Here we report our detailed investigation of intact phosphoprotein enrichment using Ti4+‐IMAC. With BSA and standard intact phosphoprotein β‐casein, selectivity with a wide range of mixing ratios of 1:1, 1:100, 1:500, 1:1000 and 1:2000 (β‐casein:BSA, w/w) and binding capacity were investigated; the enrichment capability of Ti4+‐IMAC was further investigated with real biological systems, including non‐fat milk, egg white and swine liver tissue extract. The results show that Ti4+‐IMAC is a promising material for intact phosphoprotein enrichment.