Development of Dendrimer-Based Technologies for Phosphoproteomics and Glycoproteomics in Biomarker Discovery
The dynamics of post-translational modifications (PTMs) of proteins keeps cells adapting to the ever-changing microenvironment by regulating protein structure and function in the real-time mode. Of all PTMs, protein phosphorylation and glycosylation are undoubtedly two of the most common and important PTMs and have been demonstrated to be closely linked to the onset and progression of various human diseases. Our lab has been focusing on developing novel and more efficient technologies by taking advantage of the properties of dendrimers—a soluble nanopolymer with chemically well-defined structure and functional surface groups—to facilitate the detection and quantification of PTMs using several biochemical assays and mass spectrometry-based shotgun proteomics. In chapter 2, we present a sensitive phosphorylation assay on a 96-well microplate to determine total protein phosphorylation level of complex protein samples calibrated to a phosphoprotein standard. The core of the assay is a reagent termed pIMAGO that is multi-functionalized with titanium ions for its superior selectivity towards phosphorylated proteins and with fluorophores for quantification. We demonstrated the excellent specificity, sensitivity, and quantitative nature of the assay with both standard proteins and whole cell lysates. The method was then employed to measure the overall protein phosphorylation level of human cells under different treatments. At last, we investigated the practicability of the assay to serve as a sensitive tool to estimate the amount of phosphorylated samples prior to a mass spectrometry-based phosphoproteomic analysis. In chapter 3, we introduce the multiplexed quantitation of phosphorylation and protein expression based on pIMAGO. The nanopolymer allows for direct competition for epitopes on proteins of interest, thus facilitating simultaneous detection of phosphorylation by pIMAGO and total protein amount by protein antibody in the same well of microplates. This new strategy allows us to measure cell signaling events by clearly distinguishing actual phosphorylation signals from protein expression changes, thus providing a powerful tool to accurately profile cellular signal transduction in healthy and disease cells. In chapter 4, we devise a completely novel platform to capture and profile glycoproteome, termed polymer-based reverse phase GlycoProtein Array (polyGPA). Nitrocellulose membranes pre-coated with dendrimers functionalized with hydroxylamine groups were used to covalently capture pre-oxidized glycans on glycoproteins from whole protein extracts or biofluids. The captured glycoproteins can then be detected using the same validated antibodies as in standard reverse phase protein array (RPPA). We demonstrated the outstanding specificity, sensitivity, and quantitative capabilities of polyGPA by capturing and detecting the alpha-1-acid glycoprotein (AGP) standard as well as endogenous AGP in human plasma. We then applied the strategy to validate a panel of potential biomarkers for bladder cancer identified by performing comparative N-glycoproteomics of urinary microvesicles from healthy control volunteers and bladder cancer patients and label-free quantification.
Tao, Purdue University.
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