Identification of the Apatmer-Protein Interaction by Mass Spectrometry

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Project title

Code/計畫編號

MOST105-2113-M019-003

Translated Name/計畫中文名

開發質譜學方法以鑑定適體與蛋白質之交互作用區域

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=11888176

Year

2016

Start date/計畫起

01-08-2016

Expected Completion/計畫迄

31-07-2017

Bugetid/研究經費

1400千元

ResearchField/研究領域

化學

適體核酸具有對不同目標分子專一性結合的特性，其可藉由化學方式快速合成，且化學穩定性良好，因此具有取代抗體做為分子辨識、生物技術、或臨床治療與診斷上的潛力。近年因SELEX技術的開發，可由大量隨機合成的核酸序列中快速篩選出針對目標分子的適體核酸，因此大幅增加適體核酸的應用層面。然而，針對適體核酸與其辨識蛋白質分子間交互作用的方式，目前仍須以傳統耗時的複合體純化、結晶與X射線晶體繞射法進行。本計晝將開發以質譜技術為平台之分析方法，藉由化學交聯反應固定適體核酸與蛋白質複合體的結構，以由下而上式的蛋白質體學分析方法，以期快速地決定適體核酸與蛋白質分子的結合部位。本計晝將包含：（1)以細胞色素C與其適體核酸為模型進行分析流程開發；(2)以人類凝血酶蛋白與其適體核酸做為方法測試與驗證；(3)應用於對腫瘤細胞株具專一性之適體核酸，可決定其辨識蛋白分子及其結合部位。本方法雖無法提供完整的複合體三維空間結構，但由於可快速鑑定出交互作用的區域，即可針對適體核酸對目標蛋白質的結合方式，從分子層級上瞭解其作用機制，此結果對於適體核酸在應用層面上的發展，乃至臨床治療與診斷上將有極大的助益。 Aptamers are oligonucleotides which can specifically recognize target molecules. Aptamer have good chemical stability and can be prepared by chemical synthesis. Therefore, aptamers have high potential to be the substituent molecules for antibody as for the purpose of molecule recognition, biotechnology application, or clinical diagnostic or therapeutic usage. Since the rapid screening method, systematic evolution of ligands by exponential enrichment (SELEX), has developed, to identify the unique aptamer for a target molecule from a pool of synthesized oligonucleotides with random sequences becomes more feasible. However, to determine the structural correlation for aptamer-protein interaction remains challenges because the current structure determination methods, x-ray crystallography and NMR, are both time-consuming and labor-intensive techniques. We hereby propose a Mass Spectrometry based method to identify the molecular interaction region of aptamer-protein complex. Briefly, the structural correlation of aptamer and protein will be fixed by chemical cross-linking reaction followed by the mass spectrometry-based bottom-up proteomics analysis. Three milestones are planned in this proposal. (1) Cytochrome c-aptamer complex will be utilized for method development. (2) The known structure of thrombin-aptamer complex will be employed for validation of this new method. (3) To identify target proteins of tumor-specific aptamer and determine their interaction structure. Although the newly developed mass spectrometry based method cannot provide the complete aptamer-protein complex structure, it provides reliable structural information about the regions how the aptamer recognizes or interacts with target protein. We believe that the application of aptamer for protein recognition would be more important in the future since their interaction mechanism has been revealed by this method.

Keyword(s)

Aptamer
Aptamer-Protein Interaction
Mass Spectrometry
適體
適體核酸與蛋白質交互作用
質譜分析

DSpace CRIS

Identification of the Apatmer-Protein Interaction by Mass Spectrometry

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