Relative Quantification of Potential Biomarkers of HCV-related Diseases by LC-MS/MS

肝癌是目前世界上盛行率第三的惡性腫瘤。慢性C型肝炎患者的肝細胞因損傷而發炎，致使纖維化與結節化，最後形成肝硬化。而70%至80%的肝癌患者併有肝硬化，顯見若能儘早檢測出肝纖維化或硬化，對於肝癌病人的早期篩檢與治療評估，有相當大的幫助。生物標誌可反映出生物體的生理狀況。傳統上偵測蛋白質量的改變主要以抗體法為主，但因為發展適合的抗體有許多限制，而且抗體法無法同時針對數個蛋白同時進行偵測，使針對具潛力的生物標誌的驗證難以進行。質譜法定量蛋白體學可做為另外一種對生物標誌進行定量的方法。本論文的研究目的是以質譜儀開發免標法相對定量的平台，檢測並驗證C型肝炎病人血清中具潛力的生物標誌。透過文獻的搜尋與Western Blot的分析，我們發現Alpha-2-macroglobulin (A2M)、Alpha-1-B glycoprotein (A1BG)及Beta-2 glycoprotein (B2G1) 在C型肝炎帶原者 (chronic hepatitis C；CH-C) 、慢性C型肝炎併肝硬化 (CHC - LC) 及慢性C型肝炎病毒相關肝細胞癌併肝硬化 (CHC-HCC-LC) 三個群組中表現量高於正常人。接著用LTQ-Orbitrap Velos，以一維電泳膠體增強-液相層析質譜法 (1D gel-enhanced LC-MS；1D GeLCMS) 定量正常人與C型肝炎相關疾病患者血清中的潛力生物標誌。藉由全質譜掃描選出最適合的母離子做為目標胜肽。之後加入外源蛋白GroEL做為內標準物，以同樣方式選出參比胜肽，校準膠內水解法過程酵素活性作用不一以及後續胜肽粹取時產生的變異。最後以選擇離子偵測模式 (Selected Ion Monitoring；SIM)對C型肝炎相關疾病患者血清進行檢測，驗證A2M及A1BG的含量在C型肝炎相關疾病患者血清中表現量較高。HCC is the third most common cause of cancer-related deaths in the world. Patients with chronic liver inflammation and injury may lead to liver fibrosis, abnormal nodule formation and finally liver cirrhosis. Cirrhosis is seen in about 70-80% of HCC. The early detection of fibrosis and cirrhosis has important clinical implications in the diagnosis、assessment and treatment. Biomarker can be used as an indicator of a biological state. Traditionally, antibody-based methods are widely used to quantify the expression level of specific protein. However, there are many limitations to develop effective antibodies and hard to develop a multiplex assay. Mass spectrometry-based quantification methods has recently emerged as an alternative method for quantitation. In this study we developed a label-free quantitative platform for selectively examining multiple medium - abundance protein levels. Alpha-2-macroglobulin (A2M), alpha-1B-glycoprotein (A1BG) and Beta-2 glycoprotein (B2G1) were selected with literature search and then validated by western blot We found the concentration of all the three proteins were higher in HCV-related disease cohorts than in the normal. We then quantitated these three proteins by 1D Gel-enhanced LC-MS with LTQ-Orbitrap Velos. The best-flyer peptides of target proteins were confirmed in full scan mode as target peptides. Then we spiked an exogenous protein, GroEL, just prior to in-gel digestion as internal standard which can adjust for the differences from enzymatic activities and sample losses during experiment. Finally we validated that the concentration of A2M and A1BG were higher in HCV-related diseases cohorts than in normal in SIM scan mode