title: Chemical visualization of phosphoproteomes on membrane.
abstract: With new discoveries of important roles of phosphorylation on a daily basis, phospho-specific antibodies, as the primary tool for on-membrane detection of phosphoproteins, face enormous challenges. To address an urgent need for convenient and reliable analysis of phosphorylation events, we report a novel strategy for sensitive phosphorylation analysis in the Western blotting format. The chemical reagent, which we termed pIMAGO, is based on a multifunctionalized soluble nanopolymer and is capable of selectively binding to phosphorylated residues independent of amino acid microenvironment, thus offering great promise as a universal tool in biological analyses where the site of phosphorylation is not known or its specific antibody is not available. The specificity and sensitivity of the approach was first examined using a mixture of standard proteins. The method was then applied to monitor phosphorylation changes in in vitro kinase and phosphatase assays. Finally, to demonstrate the unique ability of pIMAGO to measure endogenous phosphorylation, we used it to visualize and determine the differences in phosphorylated proteins that interact with wild-type and kinase dead mutant of Polo-like kinase 1 during mitosis, the results of which were further confirmed by a quantitative phosphoproteomics experiment.
authors.affiliation: Department of Biochemistry and Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Anton
authors.initials: A
authors.lastname: Iliuk
authors.forename: X Shawn
authors.initials: XS
authors.lastname: Liu
authors.forename: Liang
authors.initials: L
authors.lastname: Xue
authors.forename: Xiaoqi
authors.initials: X
authors.lastname: Liu
authors.forename: W Andy
authors.initials: WA
authors.lastname: Tao
mesh_terms: D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000032:analysis* / Q000378:metabolism; D002460:Cell Line; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D049329:Nanostructures; D010750:Phosphoproteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000378:metabolism; D020543:Proteome; D040901:Proteomics; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000378:metabolism; D000097869:Polo-Like Kinase 1
chemical_list: D000906:Antibodies; D018797:Cell Cycle Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D020543:Proteome; D011518:Proto-Oncogene Proteins; D000097869:Polo-Like Kinase 1