Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22022
標題: 基質輔助雷射脫附游離法搭配微結構濃縮晶片應用於磷酸化胜肽與黏多醣之快速檢測
Application of MALDI-TOF MS with radiate micro-structure chip in rapid screening of phosphopeptides and mucopolysaccharides
作者: 李坤頤
Li, Kun-In
關鍵字: 基質輔助;MALDI;質譜儀;黏多醣;磷酸化;晶片;MS;mucopolysaccharidois;phosphorylation
出版社: 分子生物學研究所
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摘要: 
黏多醣儲積症 (mucopolysaccharidosis, MPS) 的起因為人體負責降解黏多醣之基因發生缺陷所致,造成無法代謝的黏多醣持續在人體內如關節、組織、器官等各處大量累積。隨著酵素缺乏的程度差異, MPS 病患的病症也有所差異,但多伴隨如器官肥大、臉部畸形、多發性成骨不全 (dysostosis multiplex),以及聽力、視力,關節移動能力的受損,或是呼吸道與心血管受損等問題。於此希望建立一套搭配 MALDI-TOF MS 快速純化鑑定尿液中黏多醣的技術,使可能的病患可被提早檢測之目的。
樣品前濃縮與純化處理是最經常應用於增加MALDI-TOF MS 靈敏度的兩項方法,於此我們使用微結構晶片對樣品進行濃縮,增加 MALDI-TOF MS 偵測之靈敏度。比較相同體積的樣品與基質混合液點於傳統樣品盤與微結構晶片之分析結果,後者所形成之面積較前者為小,且訊號強度可放大 124% ~ 294% 不等。藉由衍生化步驟並使用陰離子交換樹脂、HLB 固相萃取可成功純化出尿液中之黏多醣分子,預期這些醣類分子可作為偵測不同黏多醣儲積症之生物標誌 (biomarker)。
可逆性的蛋白質磷酸化修飾作用可調控細胞中如增生、分化、轉錄等現象,而質譜儀是目前最常被廣泛用於鑑定磷酸化蛋白質的工具,藉由質譜儀不僅可找出磷酸化蛋白質,甚至其磷酸化位置也可被鑑定。由於磷酸化蛋白於生物體內的含量極低,加上直接以酵素水解後的胜肽混合物作為樣品分析,會發生離子抑制效應現象 (ion suppression) ,因此在進入質譜儀分析前,樣品之濃縮以及磷酸化胜肽之純化倍顯重要。因此希望藉由 Fe3O4 奈米磁性粒子搭配微結構晶片建立一快速且簡單的純化步驟,藉由與其他實驗步驟相較,本方法整體實驗時間只需耗費五分鐘;而利用相同濃度之樣品所獲得之實驗結果,on-chip 步驟之磷酸化胜肽訊號強度較 in-tube 步驟之訊雜比 (S/N ratio) 可高出27倍。

The lysosomal storage disorder mucopolysaccharidoses (MPS) caused by a deficiency in the activity of the lysosomal exohydrolases. Undegraed mucopolysacchrides accumulated in joints, tissues, and organs. The MPS share many clinical manifestations, including organomegaly, abnormal facial features, and dysostosis multiplex. Impaired hearing, vision, and joint mobility, as well as abnormal airway and cardiovascular function, are common, although there is wide clinical heterogeneity within each enzyme deficiency. However experiment diagnostic methods, have not be well established through MALDI-TOF mass spectrometry, allow patients to be treated earlier in the course of their disease.
Sample preconcentration and purification are most often used procedure to improve MALDI sensitivity. Herein, we used micro-structure chip for concentrating samples for MALDI-TOF MS analysis. Sample spots formed on our chip were much smaller than those on an unmodified plate with the same total volume, and significant enhanced results were obtained by MALDI-TOF MS. And signal intensity analyzed by chip was enhanced to 124% ~ 294% when compared to MALDI-TOF MS target plate. Oligosaccharides isolated from the urine of normal people using PMP derivatization, anion exchange and HLB extraction chromatography were identified using MALDI-TOF MS. We suggest that these glycosaminoglycan-derived oligosaccharides may be useful biochemical markers for the identification and the clinical management of mucopolysaccharidosis patients.
Reversible protein phosphorylation is a critical mediator of many cellular processes including proliferation, differentiation, and transcription. Mass spectrometry (MS) has recently become an important tool for characterization of phosphorylation proteins. Not only can phosphorylation be identified, but phosphorylation sites can also be assigned. However, direct analysis of phosphopeptides in crude enzyme-digested complex mixtures is problematic due to “ion suppression” effects and low abundance of phosphopeptides, so it is important to preconcentrate low concentration of samples and low abundance of phosphopeptides before MS analysis. Here we demonstrated another simple phosphopeptides purification procedures for MALDI-TOF MS analysis by using Fe3O4 as purification beads with micro-scale structure chip. Phosphopeptide enrichment was extremely rapid, taking approximately 5 min per sample when compared to other enrichment methods. Signal-to-noise ratio of phosphopeptides after on-chip procedure was twenty-seven times higher than in-tube procedure.
URI: http://hdl.handle.net/11455/22022
其他識別: U0005-1708200913574600
Appears in Collections:分子生物學研究所

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