開發標的質譜方法分析肺癌中磷酸化之訊息傳遞網絡

Targeted Mass Spectrometry Method for Phosphorylation-mediated Signaling Network Analysis and Verification in Lung Cancer

蔡亞妏(Ya-Wen Tsai)

指導教授 ： 陳玉如

國立臺灣大學/理學院/化學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.03047

摘要

近年來質譜技術發展漸趨成熟，已經可以針對生物樣品中磷酸化蛋白質進行大規模的鑑定與定量分析。然而鑑定到的蛋白質磷酸化位置與其表現量的變化，常需要在更多檢體中有更進一步驗證。在免疫學的傳統驗證方法上，針對特定磷酸化位置具有高專一性的抗體製造不易，而使得進一步的驗證遭遇到困難。因應這個問題，在本篇論文中，我們藉由多反應監測 (multiple reaction monitoring, MRM)質譜法針對幾十個到幾百個蛋白質上磷酸化特定位置進行驗證。為了兼顧定量重複性與準確性以及建立一個高通量的定量驗證方法，我們同時比較了三種MRM模式的定量效能，分別為:MRMHR, EDC-MRMHR (Enhance duty cycle-MRMHR)和 SWATHTM (Sequential window acquisition of all theoretical mass)方法。透過已知比例的標準蛋白質(GPB, ADH 和 Enolase)混入大腸桿菌 (E.coli)的全部蛋白質中，SWATHTM 不僅提供了最高的偵測靈敏度(訊號強度為其他方法的1.14到4.34倍)，也提供良好的定量準確性和精準度(< 6% CV)。此外，其定量最低極限(Limit of quantitation, LOQ) 的濃度更低於0.16fmol。並且在大規模的蛋白質體(E.coli)中高達713個的蛋白質可經由SWATHTM進行表現量的驗證。其定量結果比傳統利用一次質譜(MS-level)定量提供更高的準確性 (標準偏差為=0.38)。
在論文的第二部分，我們將結合SWATHTM與磷酸化蛋白質體學的分析方法，去研究九個肺癌細胞株的磷酸化蛋白質體。在這九個肺癌細胞株的蛋白質體分析結果中，總共鑑定到2713個磷酸化蛋白與10156條磷酸化胜肽。從定量結果去比較，先利用CL83這株野生型表皮生長因子接受體(epidermal growth factor receptor, EGFR)的細胞株當作比較母體，找出細胞株之間彼此的個體差異，並探討EGFR基因中Exon19 deletion突變且伴隨對於EGFR TKI(tyrosine kinase inhibitor)藥物有抗藥性的細胞株，其磷酸化蛋白質體的變化。我們利用生物資訊軟體進一步分析具有明顯變化的磷酸化蛋白質，比對出他們之間的相互關係以及訊息網絡，這些訊息網絡的功能與癌症的演進、細胞內的運動、細胞的型態和細胞對藥物的反應有關。經過SWATHTM方法驗證其表現量後，我們一共得到12個蛋白質，可能是造成抗藥性反應的蛋白質(CTNNB1, JUP, MORC2, EZH2, TRA2B, RAB9A, ACIN1, C11orf59, CANX, DEK, FNBP4 and GTF3C1)。蛋白質磷酸化的變異，往往會造成細胞內訊息傳遞的異常或者導致疾病。本論文所發展之定量驗證方法，提供了一個高敏度且高通量的分析定量平台；藉由野生型EGFR 與產生突變的EGFR的細胞株其磷酸化蛋白質表現的差異，可增進對EGFR突變和肺癌抗藥性的了解。

關鍵字

多反應監測 ； 蛋白質磷酸化 ； 肺癌 ； 表皮生長因子接受體

並列摘要

The developments in mass spectrometry-based proteomics enabled the identification and quantification of site-specific phosphorylation in biological samples. However, the lack of the phosphosite-specific antibody with good specificity has been the bottleneck for the subsequent validation by conventional immunoassy. Nevertheless, multiple reactions monitoring (MRM) enables highly reproducible quantitation of hundreds of phosphosites. Therefore, label-free MRM methods are selected to validate phosphoproteomics on lung cancer in this study to reveal the correlation of phosphorylation signaling networks with different phenotype of EGFR in lung cancer cells. We first evaluated the quantitation dynamic range of MRMHR, Enhance duty cycle-MRMHR (EDC-MRMHR) and Sequential window acquisition of all theoretical mass (SWATHTM). The Limit of quantitation (LOQ) of these methods is the same (0.16 fmol), yet, SWATHTM method provided highest extracted ion chromatography (XIC) area of each transition. Next, the quantitation accuracy and precision were evaluated triplicate analysis of three proteins (GPB, ADH and Enolase) spiked in E.coli. Based on the quantitation results of the three proteins, EDC-MRMHR and SWATHTM methods have better accuracy and precision than Data dependent acquisition (DDA) and MRMHR methods. On the proteome scale, the quantitation accuracy of SWATHTM (SD=0.38) is much better than the DDA method (SD=0.64). Among the four methods, SWATHTM has good quantitation dynamic range and accuracy with higher ion signal intensity.
In the second part of the thesis, we applied the SWATHTM method combining with phosphoproteomics approach to verify the phosphorylation sites in 9 lung cancer cell lines with different extent of drug resistance and epidermal growth factor receptor (EGFR) status. A total of 10156 phosphopeptides from 2713 phosphoproteins were identified by the DDA method. By the comparison between wild-type EGFR cell line (CL83) and phenotype with drug resistance to EGFR tyrosine kinase inhibitors (TKIs) in (CL25, CL100, PC9IR), up-regulated phosphoproteins were mapped to the network with function of cancer progression, cellular movement, cell morphology and cellular response to therapeutics. Through the quantitative comparison on the phosphoproteomics profiles between the drug-sensitive or resistant cell lines, 12 potential drug resistant targets: CTNNB1, JUP, MORC2, EZH2, TRA2B, RAB9A, ACIN1, C11orf59, CANX, DEK, FNBP4 and GTF3C1 were filtered and verified by SWATHTM method had revealed. The differential phosphoproteomic patterns suggest aberrant protein phosphorylation and network functioning in lung cancer with wild type EGFR and mutate EGFR. We have provided a strategy for systematic quantitation and validation of phosphoproteomics of lung cancer cells.

並列關鍵字

MRM ； phosphorylation ； lung cancer ； epidermal growth factor receptor ； EGFR

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