High-throughput Phosphorylation Screening and Validation through Ti(IV)-Nanopolymer Functionalized Reverse Phase PhosphoProtein Array

Protein phosphorylation is one of the most important and widespread molecular regulatory mechanisms that controls almost all aspects of cellular functions in animals and plants. Here, we introduce a novel chemically functionalized Reverse Phase PhosphoProtein Array (RP3A) to capture and measure phosphoproteomes. RP3A uses polyamidoamine (PAMAM) dendrimer immobilized with Ti(IV) ions to functionalize nitrocellulose membrane, facilitating specific chelation of phosphoproteins from complex protein samples on the array. Globular, water-soluble Ti(IV)-dendrimer allows the RP3A surface highly accessible by phosphoproteins multi-dimensionally and the captured phosphoproteins were subsequently detected using the same validated antibodies as in regular reverse-phase protein arrays. The novel chemical strategy demonstrated superior specificity (1:10,000), high sensitivity (fg level), and good quantitative nature (R2 =0.99) for measuring phosphoproteins. We further applied quantitative phosphoproteomics followed by RP3A to validate the phosphorylation status of a panel of phosphoproteins in response to environmental stresses in Arabidopsis.