Comprehensive Acetylproteomics

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Project title

Code/計畫編號

MOST107-2113-M019-003

Translated Name/計畫中文名

乙醯化蛋白體之全面性分析

Project Coordinator/計畫主持人

Pang-Hung Hsu

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=12669952

Year

2018

Start date/計畫起

01-08-2018

Expected Completion/計畫迄

31-07-2019

Bugetid/研究經費

1000千元

ResearchField/研究領域

化學

蛋白質乙醯化修飾是藉由乙醯轉移酶將乙醯基轉移至賴胺酸側鏈胺基或蛋白質的N端，而此蛋白質後轉譯修飾則是由去乙醯酶進行負向調控。蛋白質乙醯化修飾在細胞中具有非常重要的功能，可直接影響細胞內的基因表達與代謝調控，失去調控的蛋白質乙醯化將對細胞造成嚴重影響，如導致細胞癌化甚至引發細胞凋亡。目前乙醯化蛋白體學的研究，主要仍是採用以泛乙醯化賴胺酸抗體富集細胞中具乙醯化修飾之蛋白質或胜肽，再搭配液相層析串聯質譜法進行分析。此傳統方法僅能鑑定出含有乙醯化修飾的賴胺酸位點，但無法明瞭負責此賴胺酸上的乙醯化修飾是經由何種乙醯轉移酶或去乙醯酶所調控。為完整瞭解細胞內乙醯化的生理調控現象，本計畫分別針對乙醯轉移酶與去乙醯酶分別提出新的蛋白體分析方法，以細胞外酵素反應配合化學標記反應與質譜分析技術，達成可同時鑑定蛋白質中乙醯化位點並確認其調控酵素。本計畫之主要目標為：(1) 開發細胞外乙醯轉移酶酵素反應配合質譜分析以鑑定特定乙醯轉移酶的作用蛋白及其賴胺酸位點；(2) 開發細胞外去乙醯酶酵素反應並搭配質譜分析以鑑定特定去乙醯酶的基質蛋白及其賴胺酸位點；(3) 開發蛋白質N端乙醯化的全面性鑑定方法；(4) 建構完整的細胞內乙醯化修飾網路。本計畫成果可提供對細胞內乙醯化的調控機制進行分析，特別是在確知進行調控的乙醯轉移酶與去乙醯酶身份後，即可從分子層級探討蛋白質乙醯化修飾的生理意義，能全面性的瞭解細胞內的乙醯化調控網路。由於本計畫在去年提出時曾獲科技部提供一年期之經費補助，然而所設定之研究目標無法在一年內完成，因此本年度根據已執行計畫半年的經驗，調整後續的研究目標與實驗方法，再次提出為期兩年的延續型研究計畫，以期完成此乙醯化蛋白體的研究。Protein acetylation is one of the most important post-translational modifications in organisms. The acetyl group is transferred from acetyl coenzyme A to the amino group of lysine side chain by lysine acetyltransferase. Deacetylase, on the other hand, is responsible for the removal of acetyl group from the modified lysine in acetylated proteins. Protein acetylation is tightly correlated with gene regulation and cellular metabolism; moreover, the dysregulation of acetylation leads to serious consequences, such as oncogenesis or apoptosis. The current acetylproteomics study is based on the enrichment of acetylated proteins or peptides by pan-anti-acetyl-lysine antibody followed by the LC-MS/MS analysis. However, this common method provides limited information about acetylation such as the protein identifications and the determination of lysine sites with acetylation. The corresponding acetyltransferase as well as deacetylase related in the regulation of these acetylation are missing from this method. In order to tackle this limitation, a novel proteomics method based on the in vitro enzyme assay coupled with chemical labeling followed by LC-MS/MS analysis is proposed here. The major aims for this proposal include: (1) to develop an in vitro acetyltransferase assay for acetyltransferase-specific acetylproteomics; (2) to develop an in vitro deacetylase assay for deacetylase-specific acetylproteomics; (3) to develop a new method to identify protein N-terminal acetylation; (4) to create a comprehensive acetylproteomics interaction network based on the previous three aims. Once the connection between acetylation sites and their regulatory acetyltransferase/deacetylase is established, a comprehensive acetylation network can be constructed. We believe that the proposed approaches can help us understand detailed mechanism of acetylation from molecular level and also provide a new vision in acetylproteomics. This proposal is an extension from my last year MOST proposal since I only obtained one-year support from MOST. After carrying out the experiments in the past few months, I have revised some of the experimental design and hope to perform for a better result. Here, I proposed for a further two-year extension to accomplish this comprehensive acetylproteomics project.

Keyword(s)

Acetylproteomics
Acetyltransferase
Deacetylase
Mass Spectrometry
乙醯化蛋白體學
乙醯轉移酶
去乙醯酶
質譜法

DSpace CRIS

Comprehensive Acetylproteomics

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