New Approach for Acetylproteomics

View Statistics Email Alert RSS Feed

Information

Details

Project title

Code/計畫編號

MOST104-2113-M019-001

Translated Name/計畫中文名

乙醯化蛋白體學之方法開發

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=11483206

Year

2015

Start date/計畫起

01-08-2015

Expected Completion/計畫迄

31-07-2016

Co-Investigator(s)

Chih-Ching Huang

Bugetid/研究經費

1500千元

ResearchField/研究領域

化學

目前以全面性的方式進行乙醯化蛋白體學的研究仍具挑戰，其原因在於乙醯化的化學結構穩定，且蛋白質或胜肽上具有乙醯化修飾後不具電荷可進行分離，更重要的在於此修飾於賴氨酸側鍊的鍵結與胜肽鍵結相同，這些因素都導致鑑定乙醯化官能基技術上的障礙。在本計晝中，我們將結合新型態磁性奈米複合材料的開發、採用具選擇性且以非抗體純化的方式、以及質譜蛋白體學分析技術，開發一快速、高效率且對乙醯化修飾具高度專一性之分析方法，可應用於全面性乙醯化蛋白體學的探討。本三年計晝包含以下三個主要研究目標：1)開發磁性複合奈米材料以應用於具乙醯化修飾胜肽之富集； 2)在細胞外對分別以人工合成之乙醯化胜肽，以及細胞中萃取出之組蛋白進行乙醯化修飾胜肽之富集測試，並對所開發之磁性複合奈米材料進行改良，同時將方法進行最佳化；3)應用所開發之磁性奈米複合材料對缺氧環境下，細胞内乙醯化蛋白質體的變化進行分析。本計晝中所開發的方法，使用化學方式而非抗體方式對具有乙醯化修飾之賴胺酸進行富集，因此更適用於全面性鑑定所有蛋白質中之乙醯化修飾位置。我們期望本研究計晝能在乙醯化蛋白體學的研究上提供一嶄新且全面的方法。 Under the overall influences of global changes, ocean acidification is an inevitable trend. For marine diatoms, the impact of ocean acidification may alter their growth efficiency, and subsequently affect the transfer of organic carbon along the grazing food chain. A better understanding of diatom responses to ocean acidification is important to the evaluation and forecasting concerning fishery resources. With these considerations, the present submission proposes a 3-year study plan that aims to investigate how an acidic environment influences gene expressions in diatoms. Special attentions will be placed on responses of the phosphate uptake system. In the first part of the proposed research, culture media with predetermined pH values will be prepared according to guidelines issued by European Commission. These media will be used to culture a model diatom, Phaeodactylum tricornutum, and its transcriptomes as well as proteomes will be constructed to observe expressional variations in all genes. In the second part of this proposal, two diatom spcies, Chaetoceros affinis and Skeletonema costatum, will be used as the experimental subjects. The former species contains a type II Na+/Pi cotransporter gene only while the latter contains both a type II and a type III transporter genes. Growth parameters will be monitored to determine which species adapts better in a low pH environment. Quantitative reversetranscription polymerase chain reaction will be used to measure the expression levels of genes involved in phosphate uptake. In addition, SILAC proteomic analysis specific to transporter proteins will be conducted to quantify gene expressions at the protein level. Professor Pang-Hung Hsu of Taiwan Ocean University is invited to join the project as a co-principal investigator to provide guidance in the construction of proteomes. This proposal employed advanced techniques to study an ecologically important topic. Experimental results generated by this project will shed light on the molecular mechanisms of phosphate uptake and growth maintenance in marine diatoms under ocean acidification. Evolutionary strategies between diatom species will be compared, and implications will be made for variations in fishery resources due to ocean acidification.Even until now, the comprehensive study of acetylproteomics still remains to be challenging. Acetylation modifies lysine side chain s-amino group and it is very stable in terms of chemical property. The acetylated lysine does not contain charges on its side chain; this makes separating it by charge-difference impossible. In addition, the acetyl-linkage is identical to the amide bonding in the protein backbone structure; therefore, it is difficult to chemically distinguish lysine acetylation. In this 3-year project, we proposed new approach to identify acetylation sites based on the integration of newly developed super paramagnetic iron oxide nanomaterials and the mass spectrometry-based proteomics technique. This approach is highly specific to lysine acetylation but does not require affinity purification via acetyl lysine antibody. Three specific aims are included in this project. Specific aim 1 focuses on the development of super paramagnetic iron oxide nanomaterials, of which the surface is modified with functional group for recognition of peptides. The specific aim 2 is in vitro examination for the enrichment efficiency of acetylated peptide by the newly developed nanomaterials. The synthesized acetylated peptide and histone extracted from cells will be used as materials to evaluate the nanomaterials developed from aim 1. As for the specific aim 3, we plan to apply this new approach for comprehensive study of acetylproteomics by using cells under hypoxia condition as the test model. Since the acetyl peptide is recognized chemically in our method and no pan-specific antibodies will be used, our method will be more generic than the current strategy for acetylproteomics study. We believe that our method can provide new insights for the field of acetylproteomics in the future.

Keyword(s)

Acetylation
Super paramagnetic iron oxide nanoparticles
Mass spectrometry-based acetylproteomics
乙醯化後轉譯修飾
磁性奈米複合材料
乙醯化蛋白體質譜學

DSpace CRIS

New Approach for Acetylproteomics

Details

Description