Developing novel chemical probes for phosphoproteomics and interactomics

Linna Wang, Purdue University

Abstract

Chemical proteomics utilizes chemical probes to perturb, target, or manipulate proteomes to understand their functions at the cellular and molecular levels. Here we apply novel chemical probes to facilitate the analyses of protein modifications exampled by protein phosphorylation and to identify nanoparticle (including synthetic nanoparticle and virus particle)-interacting proteins. As one of the major tools to study proteomes in an organism, SDS-PAGE has been widely used for proteomics but there are only limited applications for phosphoproteomic studies due to the lack of technologies for specific detection of phosphoproteins in gels. In Chapter 2, we reported a strategy for simultaneous Visualization and Identification of Phosphoproteome in gels (VIPing) through coupling specific detection of phosphoproteins with protein identification and phosphorylation site mapping by tandem mass spectrometry. The core of the strategy is a novel compound multifunctionalized with a titanium ion (IV) for outstanding selectivity towards phosphorylated residues, a fluorophore for visualization, and a biotin group for phosphopeptide enrichment. The sensitivity and specificity of the VIPing strategy was demonstrated using standard protein mixtures and complex cell extracts, and the method was applied to study the phosphorylation changes of an essential tyrosine kinase Syk and interacting proteins upon B-cell stimulation. The novel technique provides a powerful platform for gel-based phosphoproteomic studies. In Chapter 3, we further developed novel chemical probes and imaging reagents that allow visualization of phosphoproteins from different samples on the same SDS-PAGE gel. The new strategy, termed differential gel electrophoresis of phosphoproteins (DIGEP), is based on a novel reagent that consists of a titanium ion (IV) for outstanding selectivity towards phosphorylated residues, a fluorophore for visualization, and a photoreactive crosslinker for covalent labeling of phosphorylated proteins. The specificity of DIGEP towards phosphoproteins and its ability to differentially detect phosphoproteins were demonstrated using standard protein mixtures and whole cell extracts. Endocytosis is one of the major mechanisms employed by organisms to invade cells and cause infectious diseases subsequently. A majority of nanoparticles and viruses enter host cells via endocytosis. Our understanding of the complex cellular entrance pathways involved in endocytosis would greatly benefit from a comprehensive characterization of the key proteins involved in this dynamic process. In Chapter 4, we chemically derivatized polyamidoamine (PAMAM) dendrimers and devised a novel proteomic strategy named TITAN (Tracing Internalization and TrAfficking of Nanomaterials) to reveal protein-dendrimer interactions in their native conditions with a time-resolved systems biology-based approach. Dendrimers functionalized with photoreactive crosslinkers were internalized by HeLa cells and irradiated at set time intervals, then isolated and subjected to label-free quantitative proteomic analysis. In total, 809 protein-dendrimer interactions were identified during dendrimer uptake, traceable to two major endocytic mechanisms (clathrin-mediated and caveolar/raft-mediated). The direct involvement of the two pathways was further established by the inhibitory effect of dynasore on dendrimer uptake and changes in temporal profiles of involved proteins. Based on the principle of TITAN, a strategy to investigate the virus-host interaction during cellular entry in their native conditions with a time-resolved systems biology-based approach was introduced in Chapter 5. Sindbis virus (SINV) functionalized with a photoreactive crosslinker and a handle for isolation was internalized by HeLa cells and irradiated at set time intervals, then isolated and subjected to label-free quantitative proteomic analysis. In addition to the observation of major components involved in endocytosis as expected, we also identified a potential new receptor for SINV internalization in HeLa cells.

Degree

Ph.D.

Advisors

Tao, Purdue University.

Subject Area

Biochemistry

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