化學蛋白質體學中所搭配的親和性層析，利用了化合物和蛋白質之間有特異性的交互作用成為一個強而有力之特定蛋白質搜尋技術。本研究目標為提出一發展方法去闡明除了FLT3-TKD之外，找到其他新的BEL-K（急性骨髓性白血病候選藥物）的目標物。我們在此建立一個高通量的方法來分析在MV4-11細胞株內與藥物具分子間交互作用之蛋白質。MV4-11是其中一種FLT3突變的細胞株，因為FLT3的信號傳導通路會被FLT3酪胺酸激脢抑制劑阻斷，以往的研究常針對FLT3開發急性骨髓性白血病抑制劑。我們使用BEL-K合成的生物素標記化合物並固定在鏈黴親合素的顆粒上來製作管柱；以蛋白質標準品和FLT3-TKD加入BEL-K管柱用以評估適當的沖洗和洗脫條件。MV4-11細胞株裂解液被當作研究來源，以BEL-K的親和性層析研究其目標蛋白質。然而被洗脫的蛋白質進一步送進凝膠電泳（SDS-PAGE）和液相串聯質譜（LC-MS/MS）分析和資料庫搜尋鑑定BEL-K標的候選蛋白質。在複雜混合物測試結果中，從BEL-K管柱的洗脫液看出，FLT3-TKD可以明顯的被富集。MV4-11細胞株蛋白組內總共有969個蛋白質在此被鑑定到。BEK-K管柱被使用來抓取MV4-11裂解液中標的候選蛋白質。有280個目標蛋白質被鑑定到；它們大部分是BEL-K的新奇作用物和潛在的目標蛋白質。藉由GeneGo路徑分析，一部份蛋白質被認為是藥物目標物而且它們和DNA的衰亡有關係。除此之外，也共同促成影響MV4-11細胞增殖原因。

Affinity chromatography of chemical proteomics is a powerful screening technique that is based on the specific interaction between compound and target proteins. The challenge is to develop methods capable of accurately elucidating novel targets of BEL-K (an acute myeloid leukemia drug candidate) beside FLT3-TKD. A platform for high throughput analyzing drug-binding protein of MV4-11 cell line is developed in this study. Mutation of FLT3 in AML is the reasons for the great interest currently shown in the development of FLT3 kinase inhibitors. MV4-11 is a FLT3 mutation cell line, thus its major signal transduction pathway could be interrupted by tyrosine kinase inhibitor. The synthesized biotin-tagged BEL-K was immobilized on streptavidin beads to make a column; standard proteins and FLT3-TKD were applied to BEL-K column for the evaluation of appropriate wash and elute conditions. MV4-11 cell lysate was served as the source for the study of protein target of BEL-K using this biotin-tagged affinity chromatography strategy. The eluted proteins were further subjected to SDS-PAGE, LC-MS/MS analysis and database search for the identification of BEL-K target candidates. There were a total of 969 proteins identified in MV4-11 cell lysate proteome. BEL-K immobilized column was used to capture target protein candidates in MV4-11 cell lysate. Two hundred eighty target proteins were identified; most of them are novel interactors and potential targets of the BEL-K. By GeneGo pathway analysis, some of these proteins are considered as drug targets and they related to DNA apoptosis. Besides, they are also co-contributed to the effect on MV4-11 cell proliferation.

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