臺灣博碩士論文加值系統

磷酸化是常見且相當重要的蛋白質修飾，這類修飾對於細胞中蛋白質的功能和調節有重要的影響，通常發生於酪胺酸，絲胺酸和酥胺磷上。然而除了磷酸化酪胺酸有適當抗體可作為分析的工具之外，目前因為缺乏有效而直接的分析方法，所以對於絲胺酸和酥胺酸磷酸化全面性的分析並沒有太多的成果。最近，許多利用化學修飾的方法來選擇性地純化絲胺酸/酥胺磷酸化蛋白質的方法被提出來。在我的實驗中我有效地利用金屬親和性層析法進行β-elimination/aminoethanethiol addition來分離出絲胺酸/酥胺磷酸化胜肽並以質譜儀正確地鑑定出胜肽上有絲胺酸/酥胺磷酸化的位置。 利用這一技術可以正確找出合成的酥胺磷酸化胜肽和阿爾發-酪蛋白上有磷酸化的絲胺酸/酥胺磷。除此之外，利用這方法也可找出p53和Pum2H在胞外被aurora kinase A磷酸化後產生磷酸化絲胺酸的位置。同理，亦可將β-elimination/biotin tagging配合上二維電泳將有絲胺酸/酥胺磷酸化蛋白質純化出來並分析其表現量的不同。利用此方法我分析了RK3E-RCAN和v-Src transformed RK3E中絲胺酸/酥胺磷酸化蛋白質表現的不同模式。其中因v-Src而有表現量不同的蛋白質將進行質譜儀的鑑定。在比較二維電泳膠片後，總共找到有15個有差異的蛋白質。其中Annexin I已知是Src的受質，而它的功能受到其胜肽中絲胺酸，酥胺磷和酪胺酸是否磷酸化的調節。所以，利用這一技術可以純化絲胺酸/酥胺磷酸化的蛋白質並分析它們在兩種狀態下的表現量。而這些訊息將有助於我們了解有那一些蛋白質的功能會受到它磷酸化狀態的影響。在這一些找到受v-Src轉化而表現有差異的蛋白質則需要更多的實驗來證明它們在細胞中的角色以及磷酸化對它們功能的影響。

Phosphorylation is among the most common and important protein modifications which are crucial for protein functions and regulations in cellular processes. Currently global analysis of serine/threonine phosphorylation remains largely unexplored due to lack of direct analysis tools. A number of chemical modification techniques were recently reported to selectively enrich serine/threonine phosphorylated proteins. In my work, application of on immobilized metal affinity chromatographyβ-elimination/aminoethanethiol addition to enrich phosphopeptides from peptides mixtures and determine the definite sites of phosphorylation are established. Through this approach, the phosphorylation sites of synthetic phosphopeptide and ��-casein are definitely identified. In addition, the sites of p53 and Pum2H subjected to in-vitro phosphorylation by aurora kinase A are also identified. This approach can be a tool to enrich the phosphopeptides from peptide mixture and definitely determine the serine/threonine phosphorylation site. Alternatively, β-elimination/biotin tagging can be combined with the two-dimensional gel electrophoresis to enrich and characterize the serine/threonine phosphoproteins. The serine/threonine phosphorylation profiles of RK3E-RCAN and v-Src transformed RK3E cells were established through this approach. Several proteins with differential phosphorylation status related to v-Src-induced transformation were further identified by mass spectrometric analysis. Totally, 15 differential expressed proteins in 2D-gel were identified and several of them have known serine/threonine phosphorylation site. Among the identified differential expressed proteins, Annexin I is a known substrate of Src. The function of Annexin I is related to its serine, threonine, and tyrosine phosphorylation sites. Through the chemical tagging approach, the serine/threonine phosphoproteins can be enriched and characterized. This information gives us clues to that which proteins were subjected to the serine/threonine phosphorylation regulation. Further studies are needed to reveal the function of individual protein and the effects of its phosphorylation in the v-Src transformed cells.

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