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dc.contributor.author許馨云en_US
dc.contributor.authorHSU HSIN-YUNen_US
dc.date.accessioned2014-12-13T10:42:21Z-
dc.date.available2014-12-13T10:42:21Z-
dc.date.issued2011en_US
dc.identifier.govdocNSC99-2113-M009-016-MY2zh_TW
dc.identifier.urihttp://hdl.handle.net/11536/99108-
dc.identifier.urihttps://www.grb.gov.tw/search/planDetail?id=2216901&docId=354968en_US
dc.description.abstract泛素化（ubiquitination）是一種蛋白質轉譯後的修飾作用，原本被�為僅是細胞內一種 負責蛋白質�解的信號，��此�程來控制細胞中關�信號蛋白和調節蛋白的濃度，� 時�以去除�多����折疊錯誤或�變的蛋白質。但目�越來越多的研究指出這種修 飾作用在細胞中還�用來調節許多��的生物途徑以維�正常生�功能的�作，若此種 修飾作用發生�題，將�能導致疾病，例如人類退化性的神經疾病�細胞凋亡��疫(如 NF-B)與發炎�應�細胞週期�DNA的修補和抑癌基因(如p53)等，都被發�與泛素的 修飾作用有關。因此，發展高通�方法用以有效的分�這些�到精細調控的訊�傳導網 絡，找到�此修飾作用影響的蛋白質分�，將有助於疾病診斷試劑的開發。質譜技術是 目�用以分�泛素化蛋白質分�最具潛力的方法。然而，許多目�在此領域的研究�� �在定性的分�這些修飾作用。蛋白質分�的確�也�然需�相當�複的實驗�程。此 外大多數生物體內細胞的應答是������形��程度的泛素修飾，因此，建立一個 能夠定�分�這些修飾作用的方法是必�的。 本計畫將��質譜技術與��懸浮微陣列發展一高通�的泛素化蛋白質分�分�平� 並使這些候�目標分�能夠有效的大�模定�分�確�。�特定泛素��形�有親和性 的蛋白質����（ubiquitin binding domains, UBDs)將作為泛素化蛋白質分�的�步分 類之質譜增�(enrichment)工具，使這些泛素修飾的蛋白質分�根據����形�而能夠 分別的平行分�並研究�����能�應的相關功能。轉錄因�NF-kB�與在許多生� 功能�作包括�疫發炎�應�細胞的增生分化與癌化等。泛素修飾作用在其訊�傳導調 節上扮演關�性的角色並且已廣泛被研究。本計畫將以NF-kB作為模�分�以此訊�途 徑來驗證此分�方法的�行性，候�目標分�將����懸浮微陣列作有效確�。 在此為期三年的計畫中，待完�的工作分列如下： 第一年：特定泛素��親和性分�篩�與評估 建立pull-down assay與實驗�件之最佳化 第二年：建立轉錄因�NF-kB細胞內活化模� 細胞培養與泛素化蛋白質樣本製備 親和性液相層�質譜分� 第三年：整���懸浮微陣列平�： 候�目標分�確� 設計微陣列檢測�程與標的組�以建立診斷應用工具zh_TW
dc.description.abstractPost-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 toolsen_US
dc.description.sponsorship行政院國家科學委員會zh_TW
dc.language.isozh_TWen_US
dc.title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zh_TW
dc.titleDevelopment of a Proteomic Approach Combining Suspension Microarray to Enable the Profiling and Multiplexed Validation of the Ubiquitin-Associated Interactomeen_US
dc.typePlanen_US
dc.contributor.department國立交通大學應用化學系（所）zh_TW
顯示於類別：研究計畫


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