陳玉如陳頌方Chen, Yu-JuChen, Sung-Fang謝宜庭Hsieh, Yi-Ting2020-10-19不公開2020-10-192019http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22G060642022S%22.&%22.id.&amp;http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/111096肝癌在癌症中致死率為第三名。然而，目前診斷肝癌的工具的靈敏度及專一性有限。甲型胎兒蛋白是目前臨床上常使用的生物標記蛋白，然後在40%的肝癌病患濃度卻是比較低的 (2 ng to 20 ng/mL )，造成誤判此需要一個好的血清標記作為肝癌的診斷，特別是針對肝癌初期的病患，因為在早期癌症是沒有症狀的。 蛋白質的醣基化修飾的疾病生物標的已廣受積極探討，因為醣基化的表現量與其結構異質性之變異已與包含癌症等幾種疾病有關。此研究中，我們嘗試針對肝癌（HCC）的生物標的蛋白紅血球結合蛋白 (Haptoglobin，Hp）中的位點特異性糖基化進行量化。我們的團隊於先前已經利用非靶向質譜方法（untargeted-MS）和生物資訊分析找出可能在肝癌患者中具有表現上升的 19 個Hp 特殊醣型。為了驗證這些可能的醣胜肽，在此論文第一部份，我們開發了一種奈米探針結合質譜的技術，平行反應監測（PRM-MS），以應用於這19 個醣胜肽依據不同的醣胜肽修飾 (價數，氧化，鈉的加合物) 衍生出49個衍生物醣胜肽的定量。利用以血紅蛋白修飾的磁性奈米探針（MNP@Hb）從血清中純化出 Hp，經由酵素水解消化後，再進行親水性作用層析法 (Hydrophilic interaction chromatography，HILIC) 將醣胜肽做進一步分離與濃縮。在質譜技術方面，為了減少干擾物的干擾，每條醣胜肽都有最佳的分離窗口， 有干擾勿干擾的醣胜肽前驅物就會選擇狹小的分離窗口 0.7 Da 到 1.2 Da； 沒有干擾勿干擾的前驅物就會選擇1.4 Da 的分離窗口。在此論文的第二部分，將此方法應用於 51個肝炎，54個肝硬化、34個低濃度甲型胎兒蛋白 (20 ng/mL)的肝癌和17個高濃度甲型胎兒蛋白(>20 ng/mL)。利用SDS-PAGE及銀染，來檢測血紅蛋白修飾的磁性奈米探針（MNP@Hb）對紅血球單白標準品的純化效率，其純化效率約為85% 到100%。血紅蛋白修飾的磁性奈米探針2的最大純化量是 5μg。在醣胜肽的平行反應監測質譜法定量的方面，是利用碎片離子的總和及使用內標準品 (SGP) 校正醣胜肽含量。在這些疾病群中，有條醣胜肽在位點N184上的兩個分支含有核心岩藻醣基化和雙唾液酸的醣型 (GP15, m/z: 1045.43 (4+), A4G5F1S2 醣型)，在肝癌中的含量明顯上升 (p value : 0.064)。有三條在位點N184的醣胜肽 (GP13、GP40、GP47)，在高濃度甲型胎兒蛋白是明顯上升的 (p value 分別為0.031、0.034、0.0342)。在未來我們希望這四個可能的醣胜肽可以與其他標記蛋白像是甲型胎兒蛋白，提供肝癌診斷的平台 。Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. Serum diagnosis using alpha fetoprotein (AFP) is the current diagnosis tools for HCC. However, 40% of HCC patients have low concentration (2-20 ng/mL) in serum, which is the major challenge to cause false negative diagnosis in HCC. Consequently, robust serum markers are needed for diagnosis, especially for early-stage HCC lacking specific symptoms. Altered protein glycosylation is extensively reported to be associated with cancer initiation and progression and is emerging as potential biomarker for cancer. Previously, our group has identified 19 site-specific glycopeptides in haptoglobin (Hp), a liver-secreted protein, that are elevated in serum of HCC patients compared to high-risk groups by label-free quantitation using mass spectrometry (MS) analysis. In this work, we aim to validate the expression level of site-specific glycosylation in Hp from viral infection hepatitis (HBV and HCV) liver cirrhosis (LC), HCC-Low AFP (AFP<20 ng/mL), and HCC-High AFP (AFP <20 ng/mL) patients. To explore the alteration of these 19 site-specific glycopeptides as a potential biomarker for HCC, the hepatitis and LC groups were employed as non-cancer controls. We developed a nanoprobe-based assay coupled to parallel reaction monitoring mass spectrometry (PRM-MS) for multiplexed quantification of the 19 glycopeptides that can complement AFP to distinguish non-cancer and cancer for early diagnosis of liver cancer. In the first part of thesis, we establish and optimize the PRM-MS platform for quantitation of 49 glycopeptide precursors derived from 19 glycopeptides with different forms (charge state, oxidation, sodium adducts). The Hp was purified from serum using MNP@Hb magnetic nanoprobes and its glycopeptides were enriched using hydrophilic interaction chromatography (HILIC). To reduce interferences from co-elution of other ion with similar m/z to the target glycopeptide precursor, the PRM-MS method was optimized by setting variable isolation window size for each precursor. For the precursor with interference, the window size were set with narrow range 0.7 to 1.2 Da. For precursor without interference, the window size were set at 1.4 Da. In the second part of thesis, we applied the PRM-MS method to quantitatively compare the Hp glycopeptides in patients with hepatitis patients (n=51), liver cirrhosis (n=54), HCC-low AFP (AFP ≤ 20 ng/mL, n=34), and HCC-high AFP (AFP > 20ng/mL, n=17). The Hp purification efficiency for standard Hp which was evaluated by SDS-PAGE and followed by silver stain, were range from 85% to 100%. The maximum carrying capacity of the MNP@Hb is 5μg. The quantitation of gycopeptides was performed with sum of peak area fragment ions by using PRM-MS and normalization by SGP to eliminate the batches effect. Among the liver disease groups, a core-fucosylated biantennary glycopeptide on site N184 (GP15, m/z: 1045.43 (4+), with A4G5F1S2 glycan) demonstrated statistically significant higher level in HCC compare to non-liver cancer groups (p value : 0.064 for HCC-L and non-cancer group). In addition, 3 glycopeptides on site N184, including fucosylated trisialylated glycopeptides (GP13, m/z: 1041.18 (4+), with p value 0.031, and GP40, m/z: 1387.90 (3+), with p value: 0.034) and bisected glycopeptides (GP47, m/z: 1587.64 (3+) with p value: 0.0342), were significantly higher in HCC with high AFP concentration. In the future, we hope to add these 4 glycopeptides with other potential biomarkers for HCC, such as AFP and AFP-L3 to form a multimarker panel to further improve HCC diagnosis.肝癌紅血球結合蛋白(Hp)平行反應監測質譜法 (PRM-MS)Hepatocellular carcinoma (HCC)Haptoglobin (Hp)Parallel reaction monitoring mass spectrometry (PRM-MS)