HeLa細胞與小鼠腦中精胺酸甲基接受蛋白之蛋白體研究

Proteomic study of Methylaccepting Proteins in HeLa Cells and mouse brains

洪健仁(Chien-Jen Hung)

指導教授 ： 李娟

中山醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

蛋白質精胺酸的甲基化是一常見的轉譯後修飾，此反應是由精胺酸甲基轉移酶所催化進行。在之前的研究中顯示，精胺酸甲基接受蛋白多為鹼性的核酸結合蛋白，並且擁有arginine 與glycine rich (GAR) motifs。為了分析精胺酸甲基接受蛋白，我們使用了蛋白體 (Proteomic)分析的技術，使用二維膠體電泳 (2-dimensional gel electrophoresis)、色層焦集法 (chromatofocusing) 的管柱及MALDI-TOF-MS的分析，對這些蛋白作進一步的鑑定。首先將HeLa細胞萃取蛋白在進行二維膠體電泳後，用可以偵測精胺酸上甲基化的抗體 (7E6 , Abcam) 進行西方點墨法分析，將二維膠體電泳蛋白染色結果與西方點墨法的訊號二者進行比對，根據比對的結果，取有甲基化反應的蛋白點，使用蛋白內切酶作in gel digestion，再將產物進行MALDI-TOF-MS的分析，之後以線上軟體 (Mascot) 分析質譜數據，至資料庫中鑑定這些蛋白。這些鑑定出來的蛋白，除了hnRNP A2/B1、hnRNP A1、hnRNP G、Sam68及FUS gycline rich protein，這些已知的甲基接受蛋白外，還包括hnRNP M和54 kDa nuclear RNA- and DNA-binding protein (p54nrb) 這二個可能的甲基接受蛋白，其中的hnRNP M我們進行後續的分析，結果顯示其可能是甲基接受蛋白，但結果並不一致，還有待進一步的實驗分析。另一方面，為希望有較大量的蛋白質樣本以利後續分析，我們用液相層析法PH 9-6的色層焦集法管柱 (Mono P管柱) 去分析鼠腦組織萃取液，希望能將鹼性的核酸結合蛋白分離。發現只有少數的蛋白被分離出來;因而進一步使用核酸酶 (nuclease) 去處理鼠腦組織萃取液，使鹼性的核酸結合蛋白能夠釋放出來，再經由Mono P管柱分析。我們發現經由核酸酶處理與否之鼠腦組織萃取液，二者蛋白在Mono P管柱的fractions分佈情形是不同的，主要的差異在於核酸酶經過處理後，在管柱的flowthrough fractions蛋白是明顯增加。將這些蛋白作MALDI-Q-TOF spectrometry的分析，我們驚訝的發現主要的蛋白，大多為一些重要的代謝酵素，如: glyceraldehydes-3-phosphate dehydrogenase、malate dehydrogenase 及 transketolase，然而，並沒有含有GAR motif的核酸結合蛋白被發現。

關鍵字

精胺酸甲基化 ； 蛋白質體學 ； 甲基接受蛋白

並列摘要

Protein arginine methylation is a posttranslational modification catalyzed by protein arginine methyltransferase (PRMT). Previous investigations indicated that most of the methylaccepting proteins are basic RNA binding proteins containing arginine and glycine rich (GAR) motifs. In order to analyze the arginine methylaccepting proteins, in this study we used proteomic approaches including two-dimensional (2-D) electrophoresis, chromatofocusing chromatography (Mono P column) and MALDI-TOF-MS analysis to identify these proteins. In the lst part, HeLa cell extracts were separated by the 2-D electrophoresis and then detected by mono- and di-methylarginine specific antibody 7E6. We compared the patterns of the protein stain and the western signals and found some matched spots. The spots were cut, endoprotease in-gel digested, the fragments were analyzed by MALDI-TOF-MS and MS/MS. We identified the spots by the help of online programs (Mascot). The identification of the previously reported methylarginine-contains protein such as hnRNP A2/B1, hnRNP A1, hnRNP G, Sam68 and FUS gycline rich protein indicated our approach is feasible. In addition, our data suggested hnRNP M and 54 kDa nuclear RNA- and DNA-binding protein (p54nrb) are putative methylaccepting proteins. We got further evidence of the methylation of recombinant hnRNP M and the peptide with the RGG sequence in the hnRNP M protein. In the 2nd part, we fractionated mouse brain extracts by a chromatofocusing Mono P column with a pH 9-6 gradient. As few proteins were eluted, we further treated the brain extract with nuclease before chromatofocusing to release basic nucleic acid binding proteins. Distinct patterns of the elutted proteins in Mono P column fractions with nuclease or not were detected and the proteins in the flowthrough fractions of the column increased significantly upon nuclease digestion. The highly basic proteins released by nuclease treatment were identified by MALDI-TOF spectrometry. Surprisinly, the majority of the proteins are enzymes in important metabolic pathways such as glyceraldehydes-3-phosphate dehydrogenase, malate dehydrogenase and transketolase. However, no GAR containing nucleic acid binding proteins were identified in the basic proteins.

並列關鍵字

arginine methylation ； Proteomic ； methylaccepting Proteins

參考文獻

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