發展一結合流式微陣列之蛋白質體分析方法以作為泛素(Ubiquitin)相關之生物分子交互作用確認平台

Abstract

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Post-translational modifications (PTMs) of proteins by the covalent conjugation of ubiquitin (also called ubiquitination), which originally implicated as a signal for proteolytic degradation, have now been discovered to regulate diverse biological processes. Considering the importance of ubiquitination in normal cellular physiology, and their roles in disease etiology and progression, it will be critical to develop high-throughput methods capable of mapping the delicate networks to efficiently identify proteins modified by these small molecules. For the characterization of ubiquitinated proteins, the mass spectrometry offers great promise. However, much of current analysis focused on identifying modifications, rather than on quantifying them. The laborious validation procedure for the identified targets is another bottleneck. In addition, biological effects are often due to changes in the linkage types and the level of the ubiquitination, and thus there is intense necessity in the improvement of the methodology in the quantitative study of these modifications. This proposal aims to develop an approach combining mass spectrometry and suspension microarray for ubiquitin- modified proteins profiling and allows high throughput quantitatively validation of the candidates. The selective ubiquitin binding domains (UBDs) specific to individual types of linkages will be applied for pull down assays as an initial tool for cataloging ubiquitome profiling to discover protein panels with corresponding ubiquitin linkages and the associated functions. The nuclear factor B (NF-kB) signaling will be used as a model pathway to demonstrate the feasibility of the approach and the identified molecules will be used as the target proteins for the effective validation by the suspension microarray. In this three-year project, it is anticipated that the following work will be accomplished: (1) Selection of the affinity binders, setup and the optimization of the pull down assay (2) Establishment of the NF-kB activation protocol as a model pathway and the setup of affinity-based LC-MS/MS to demonstrate the feasibility of the approach to profile the ubiquitin-associated interactome (3) The prototype of the suspension microarray panel to enable the multiplexed validation of the hits and its potential applications as diagnostic tools