索拉非尼 (拜耳)、舒尼替尼 (輝瑞) 及臨床試驗中藥物AV-951 (樂騁) 均為小分子口服藥物，為血管內皮生長因子 (VEGF) 受體酪胺酸激酶的抑制劑，其中Sorafenib以及Sunitinib已被核准治療腫瘤生長以及血管新生。本實驗運用同重元素相對和絕對定量 (iTRAQ) 試劑搭配質譜分析的技術，針對三種不同的抗癌藥物投藥於人類肝腫瘤細胞株 (PLC/PRF/5) 的蛋白質體研究。為了降低樣品分析時的複雜度，提升低含量蛋白質/胜肽被鑑定到的機會，iTRAQ標定的胜肽樣品會先經由等電點聚焦分離儀 (OFFGEL fractionator)、強陽離子交換層析法 (SCX) 以及鹼性逆相層析法 (bRP) 作第一維分離，爾後再進行奈米級液相層析連接質譜儀進行分析，於生物性重複實驗之中，共鑑定到2010個蛋白質以及11233個不重複的胜肽。從鑑定到蛋白質總數之百分比評估，等電點聚焦分離儀、強陽離子交換層析法以及鹼性逆相層析法分別鑑定到80%、30%以及81%的蛋白質。相較於只使用鹼性逆相層析法，增加兩種分離方法可多鑑定到23%的蛋白質。索拉非尼與其他兩組抗癌藥物相比之蛋白質，使用GeneGO生物資訊軟體進行預測，挑選與神經退化疾病相關的蛋白質，利用定量聚合酶連鎖反應進行確認。其中，細胞間黏附因子1 (intercellular adhesion molecule 1, ICAM-1) 使用定量聚合酶連鎖反應及西方墨點法的驗證結果與本實驗iTRAQ上調定量結果一致，其功能為穩定細胞-細胞間的作用，以及促使白血球內皮細胞的輪迴，於病理發炎及免疫相關疾病扮演重要角色。

Sorafenib (Bayer/Onyx), sunitinib (Pfizer) and av-951 (Aveo) are oral small molecular VEGF receptor tyrosine kinase (RTK) inhibitors, and two of them are approved to treat anti-angiogenesis. In this study, we used isobaric tags for relative and absolute quantitation (iTRAQ) to investigate the protein profiles in PLC/PRF/5 cell lines treated with these three anti-cancer drugs (sorafenib, sunitinib, av-951). In order to reduce sample complexity, iTRAQ labeled tryptic peptides were fractionated by solution isoelectric focusing (sIEF), strong cation exchange chromatography (SCX) or basic reverse phase chromatography (bRP), followed by nano-LC tandem mass spectrometric analysis. A total of 11233 unique peptides were identified which were associated with 2010 proteins in two biological replicate experiments. The solution-IEF, SCX and basic RP methods permitted a total of 80%, 30% and 81% of proteins to be identified respectively. The results were complementary, and allowed more than 23% more of proteins to be identified, when compared with basic reverse phase chromatography alone. Among them, differentially expressed proteins were selected for GeneGO analysis. Proteins that associated with neurodegenerative diseases are selected for other verifications. Intercellular adhesion molecule 1 (ICAM-1) plays an important role in the pathogenesis of inflammatory or immune-related diseases, had shown up-regulated results consistently with iTRAQ, RT-PCR and western blotting.

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