穩定同位素標定之氨基酸於果蠅細胞培養方法之最佳化暨質譜定量蛋白質體學之應用

Abstract

以質譜為平台的定量化磷酸蛋白質體學研究能夠有效幫助理解細胞訊號傳遞路徑及其相關疾病。穩定同位素標記氨基酸細胞培養方法(SILAC)，是一個定量方法學中，在哺乳類細胞內標定特定氨基酸到所有蛋白質的強大定量方法。此平台並於 2008年已應用於果蠅S2細胞。 據該篇研究中指出，在配製的SILAC培養液過程中，因為血清與酵母菌萃取物必須經過透析的步驟，而此透析過程當中會去除低分子量的分子，造成S2細胞的生長速率降低。此外，由於SILAC實驗需要大量的穩定同位素標記的離氨酸和精氨酸，再加上同位素標記的氨基酸價格非常昂貴，因此使用如此大量的穩定同位素標記氨基酸是不符合經濟效益的。 在這篇論文中，我們進行了改進 SILAC系統的工作，並且克服了上述缺點。據推測，延遲細胞的生長的其中原因是因在酵母菌萃取物和血清的透析過程中流失維生素所導致。因此，SILAC培養液中添加維生素的方案，大大提高了細胞的生長速率。此外，我們降低並且最佳化了穩定同位素標記的離氨酸和精氨酸的用量而達到與前述系統一樣之標定效率，從而大大降低了成本。 使用我們改進的 SILAC配方，即添加維生素和更使用少量的氨基酸，我們定量了在Calyculin A (蛋白磷酸酶1 /蛋白磷酸酶- 2A的抑製劑) 存在以及不存在的情況下S2細胞的磷酸化蛋白質的比較。施加Calyculin A的細胞其磷酸化程度大幅增加，在沒有任何預先分餾步驟，我們一共定性了1679個第一級磷酸化位置，而當中共有470個磷酸化位置的磷酸化程度增加了兩倍以上。 由於以SILAC平台為基礎的定量蛋白質組學已被越來越廣泛地應用於細胞相互作用以及信號傳遞網絡的研究上，而標定的成本和效率，以及細胞成長速度在此平台是一個非常重要的課題。因此，我們的高成本效益和高效率的最佳化SILAC-S2平台可廣泛應用於果蠅系細胞株，為定量研究平台提供一個更可以接受的成本考量。

Mass spectrometry based quantitative phosphoproteomics research is capable of understanding cell signaling pathways and its related diseases. Stable Isotope Labeling by Amino acids in Cell culture, SILAC, is a recently developed method for quantitative proteomics facilitated by in vivo incorporation of specific amino acids into all mammalian proteins. While it is mainly used for analysis of yeast and mammalian cells in culture, this platform has also been extended to Drosophila melanogaster S2 cells in 2008 by Mann et al. It was found in that single study that the growth rate of S2 cells was reduced due to the removal of low molecular weight molecules during the process of serum and yeastolate dialysis in preparing the SILAC media. Furthermore, since isotope-labeled amino acids are highly expensive, and a great amount of stable isotope labeled Lysine and Arginine was needed, the cost of SILAC for S2 cells appeared to be economically inefficient. In this thesis, work, an improved SILAC system has been developed to overcome the shortcomings described above. It was reasoned that lack of vitamins in dialyzed yeastolate and serum would cause a delay in cell growth. Thus, the SILAC medium is supplemented with vitamin solution that significantly improved the cell growth compared to previous report. Moreover, optimized and reduced amounts of stable isotope labeled Lysine and Arginine were used to achieve the same incorporation level, thus reducing the cost considerably. Using our improved SILAC formula, with the presence of vitamin solution and less heavy amino acids, the phosphoproteome of S2 cells was quantitatively mapped in the presence and absence of added Calyculin A, a protein phosphatase-1 / protein phosphatase-2A inhibitor. Phosphorylation levels of cells treated with Calyculin A have significantly increased compared to the control. Without any pre-fractionation steps, 1679 class I phosphosites have been identified, of which 470 sites were up-regulated more than 2-fold. Since SILAC-based quantitative proteomics has been increasingly applied to discover cellular interacting or signaling networks, the cost and efficiency in either labeling or growth rate of such platform is a very important issue. Thus, our improved SILAC formula for Drosophila melanogaster cell lines will provide a more economical and hence efficient platform for all future quantitative studies.