LAUSR

Selective separation and enrichment of phosphoproteins are essential for understanding their important functions in almost all cellular processes. Here, taking advantage of the feature that cadmium ion (Cd2+ ) has an overwhelming preference for phosphates, we developed a robust and simple Cd2+ co-precipitation strategy for the selective isolation of intact phosphoproteins. After evaluating the feasibility of Cd2+ in phosphoprotein precipitation, we compared the washing protocols for the remove of non-specific binding proteins, and then used the best-performing protocol for the isolation of phosphoproteins from different complex samples. It was found that phosphoproteins can be specifically enriched from artificial protein mixtures containing α-casein, β-casein and BSA or plasma, in which BSA or plasma were served as interferences with very high molar ratios. Applying this method to enrich phosphoproteins from complex cell lysates, a high specificity was confirmed by western blotting analysis with a phosphoprotein-specific kit. Finally, we successfully applied this method to the purification of caseins from drinking milk, highlighting its potential application in the studies where purified phosphoproteins were required. In a word, this Cd2+ co-precipitation method enables universal and effective capture, enrichment, and detection of intact phosphoproteins, making it a powerful tool for the comprehensive analysis of the phosphoproteome. This article is protected by copyright. All rights reserved.