質譜儀的多重反應監測(Multiple Reaction Monitoring, MRM)/選擇反應監測(Selected Reaction Monitoring, SRM)模式對小分子的定量是非常靈敏的，而目前可將此法應用在已證實可能為生物標記但無適合的抗體能使用之蛋白質和胜肽，並進一步對目標蛋白質進行相對或絕對定量，另外此分析平台也可用來偵測無法藉由質譜儀的survey scan來進行資料庫比對的微量蛋白質，故在本研究中，發展出以金奈米粒子為主的親和性純化方法，來純化雌激素受體α (Estrogen Receptor, ERα)和先前證實在轉錄調節上為ERα之共調節因子的c-Myc蛋白質，並結合質譜儀的MRM/SRM掃描模式進行偵測。設定上選擇一對代表目標蛋白質的胜肽序列和此胜肽片段所斷裂的碎片離子，稱為MRM/SRM transitions，其選擇方式是從質譜數據經由資料庫比對所得到的序列中謹慎挑選，此方法可藉由質譜儀的MRM/SRM偵測優點，將我們所選擇的MRM/SRM transitions的訊號提升3至4個數量級，最後再將選擇的胜肽序列，即母離子(Precursor ion)，利用碰撞誘導解離(Collision Induced dissociation, CID)去做targeted MS2進一步確認胜肽結構，其實驗結果為10公分的培養皿中，若長滿MCF-7乳癌細胞，大約可以定量到1.6 pmole的ERα，而在免疫沉澱樣品中也有定到c-Myc，為了進一步確認此結果，故將c-Myc所選擇的片段去做targeted MS2，以證實胜肽結構為c-Myc片段的訊號，此實驗方法可以去對MCF-7乳癌細胞中的ERα進行定量，且能偵測一起免疫共沉澱(Co-immunoprecipitation, Co-IP)下來的c-Myc進行定性，未來可再進一步針對兩蛋白質的交互作用進行探討。

Mass spectrometry-based analytical platform called multiple reaction monitoring (MRM) or selected reaction monitoring (SRM) is a very sensitive technique for the quantification of small molecules and have recently been developed for targeted proteins and peptides, which makes it possible to verify biomarker candidate proteins even if suitable antibodies are not available. This method can be used for relative or absolute quantification of candidate protein biomarkers. This method can also be used to characterize low abundant proteins, which cannot be detected by the survey scan. In this study, a reliable SRM/MRM method for target proteins coupled with a gold nanoparticle based affinity purification (AP) methods were developed to characterize two nuclear proteins estrogen receptor α (ER-α) and c-Myc protein which was previously identified to be a co-regulator of ER-α in transcriptional regulation. For the development of MRM/SRM method, suitable sets of precursor and fragment ion masses for a given peptide, called MRM/SRM transitions, were carefully chosen to constitute definitive mass spectrometry assays that identify peptides and the corresponding proteins. The detection sensitivity of the selected ions is greatly enhanced up to 3-4 orders of magnitude by MRM/SRM method. Once a precursor is identified, the peptide structure was further characterized by targeted fragmentations through collision-induced dissociation (CID). We determined the absolute amount of ERα from a 10-cm dish of MCF-7 cells was around 1.6 pmole and the c-Myc protein was qualified and targeted by MS2 for reconfirmation. By applying this method, ER-α and c-Myc were able to be identified from MCF-7 cells and from the co- immunoprecipitation (Co-IP) which allows their interactions to be characterized.

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