Highly efficient enrichment of intact phosphoproteins by a cadmium ion-based co-precipitation strategy.

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 removal 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, ß-casein, and bovine serum albumin or plasma, in which bovine serum albumin 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.

miR-196a Upregulation Contributes to Gefitinib Resistance through Inhibiting GLTP Expression.

Tyrosine kinase inhibitor (TKI) therapy has greatly improved lung cancer survival in patients with epidermal growth factor receptor (EGFR) mutations. However, the development of TKI-acquired resistance is the major problem to be overcome. In this study, we found that miR-196a expression was greatly induced in gefitinib-resistant lung cancer cells. To understand the role and mechanism of miR-196a in TKI resistance, we found that miR-196a-forced expression alone increased cell resistance to gefitinib treatment in vitro and in vivo by inducing cell proliferation and inhibiting cell apoptosis. We identified the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) bound to the promoter region of miR-196a and induced miR-196a expression at the transcriptional level. NRF2-forced expression also significantly increased expression levels of miR-196a, and was an upstream inducer of miR-196a to mediate gefitinib resistance. We also found that glycolipid transfer protein (GLTP) was a functional direct target of miR-196a, and downregulation of GLTP by miR-196a was responsible for gefitinib resistance. GLTP overexpression alone was sufficient to increase the sensitivity of lung cancer cells to gefitinib treatment. Our studies identified a new role and mechanism of NRF2/miR-196a/GLTP pathway in TKI resistance and lung tumor development, which may be used as a new biomarker (s) for TKI resistance or as a new therapeutic target in the future.