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標題: 利用質譜技術鑑定芝麻儲存蛋白質與愛玉凝膠酵素醣基化
Determination of Storage Proteins in Sesame Seeds and Glycosylation of Pectin Methylesterase in Jelly Fig Achenes by Mass Spectrometry
作者: 蕭世良
Hsiao, Shih-Liang
關鍵字: Mass spectrometry;質譜技術;Sesame seeds;Storage proteins;Jelly fig achenes;Pectin methylesterase;Glycosylation;芝麻種子;儲存蛋白質;愛玉瘦果;凝膠酵素;醣基修飾
出版社: 生物科技學研究所
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Plant Physiol. 2003, 6, 93-105. CHAPTER 2 REFERENCES Micheli, F. Pectin methylesterases: cell wall enzymes with important roles in plant physiology. Trends Plant Sci. 2001, 6, 414-419. (2) Wen, F.; Zhu, Y.; Hawes, M. C. Effect of pectin methylesterase gene expression on pea root development. Plant Cell 1999, 11, 1129-1140. (3) Chen, M. H.; Citovsky, V. Systemic movement of a tobamovirus requires host cell pectin methylesterase. Plant J. 2003, 35, 386-392. (4) Chen, M. H.; Sheng, J.; Hind, G.; Handa, A.K.; Citovsky, V. Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement. EMBO J. 2000, 19, 913-920. (5) Hirsch, A. R.; Förch, K.; Neidhart, S; Wolf, G.; Carle, R. Effects of thermal treatments and storage on pectin methylesterase and peroxidase activity in freshly squeezed orange juice. J Agric Food Chem. 2008, 56, 5691-5699. (6) Zykwinska, A.; Boiffard, M. 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摘要: 
質譜技術被廣泛應用於現今的蛋白質研究上,除了蛋白質身份鑑定外,也應用於蛋白質轉譯後修飾作用的分析,如:磷酸化、醣基化及雙硫鍵形成等。本論文研究主題分成兩部分,主要都是利用質譜技術。第一部份是鑑定芝麻種子中主要的儲存蛋白質(11S球蛋白與2S白蛋白)在聚丙烯醯胺膠體上的相對位置,目的在於標定出可能成為過敏原的儲存蛋白質在膠體上的位置,對於未來在診斷過敏原的判斷上有所助益。第二部分是要鑑定愛玉凝膠酵素上的醣基修飾位置以及醣基結構,目的在於進一步瞭解之後,對於未來在研究醣基化與酵素活性的關係有所幫助。
在第一部分中,利用3328筆芝麻種子表現序列標幟的序列資料庫中,發現四個11S球蛋白(兩個為新發現,兩個為實驗室已發現)及三個2S白蛋白(一個為新發現,兩個為實驗室已發現)的基因。然後在實驗室得到三個新發現的儲存蛋白質同功異構型之全長基因後,本研究工作則是利用12.5%與18%聚丙烯醯胺膠體分別清晰地將11S球蛋白與2S的白蛋白同功異構型在膠體上分開,再分別利用液相層析串聯質譜儀進行身分鑑定確定在膠體上的位置,所對應是何者同功異構型。
在第二部分中,根據實驗的研究結果,發現愛玉凝膠酵素具有醣基修飾。本研究工作則是利用基質輔助雷射脫附游離化質譜儀,鑑定出在第153個胺基酸-天門冬素為凝膠酵素的醣基修飾位置。利用串聯質譜儀,分析出凝膠酵素的醣基結構以含有木糖化、無核心岩藻糖化之甘露糖或複雜形式等結構存在,並用分子模型化模擬凝膠酵素的結構,發現醣基修飾位置在酵素活性位置的背面區域。

Mass spectrometry has been nowadays extensively used in protein research, including protein identification, peptide sequencing and post-translational modification such as phosphorylation, glycosylation and disulfide bond formation. The present study consists of two parts, which were both mainly achieved by mass spectrometry. In the first part, the relative positions of major sesame seed storage proteins, 11S globulin and 2S albumin, were demarcated in SDS-PAGE. These proteins are potential allergens, and the designation thereof could be beneficial for the diagnosis of allergens in pertinent food products. In the second part, the glycosylation site and glycan structures of pectin methylesterase in jelly fig achenes are determined to provide a better understanding on the relation between glycosylation and enzymatic activity.
In the first part, two gene families separately encoding four 11S globulin (two were newly found; two were formerly published) and three 2S albumin (one was newly found; two were formerly published) were identified from a database search of 3328 expressed sequence tag (EST) sequences. The full-length cDNA sequences of the new isoforms had been completed in our laboratory. In the present work, the four 11S globulin and three 2S albumin isoforms resolved in 12.5% and 18% polyacrylamide gels were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
In the second part, pectin methylesterase (PME) in jelly fig achenes has been detected as a glycoprotein previously. In the present work, the glycosylation site was identified to be at Asn153 of mature jelly fig PME by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). The major N-glycans released from native PME were identified as xylosylated, non-core fucosylated, pauci-mannose and complex-type structures by tandem mass spectrometry (MS/MS) analyses. Molecular modeling of jelly fig PME indicated that the N-glycan was putatively attached to the back region of the active site.
URI: http://hdl.handle.net/11455/36200
其他識別: U0005-0308200916322700
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