以​多重反應監測質譜法​定量分析​肝癌​之​醣蛋白生物標​記

Abstract

基於蛋白質N型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Hp特殊醣型，為了驗證這些可能的醣型，我們開發了一種奈米探針結合質譜的技術，如多重反應監測（MRM-MS）和並行反應監測（PRM-MS），以應用於醣胜肽的定量。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。為了確保醣胜肽分析的正確性，在二次質譜的圖譜中，除了判斷來自完整醣胜肽的Y1離子（胜肽+ HexNAc）和醣碎片離子（HexNAc與HexHexNAc）以外，同時也會監測來自胜肽主鏈上斷碎的b和y離子。雖然MRM-MS可以獲得良好的線性（r2 = 0.950〜0.990）和靈敏度（62.5 ng Hp），但其偵測質量範圍使得對於檢測高分子量範圍的糖胜肽（荷質比大於1000）分析受限。因此，我們利用具有更廣泛質量範圍的PRM-MS技術，分析Hp非醣胜肽可以得到良好的線性（r2 = 0.996〜0.997）及精確度（CV = 14.7％），進一步應用此方法來定量肝臟疾病中的104條Hp醣胜肽，其中12個標的在一次質譜圖中具有良好的層析譜圖與二次質譜圖中，其醣碎片離子，呈現好的定量共析曲線。初步分析20個臨床血清樣本（各含5位具有高濃度或低濃度甲型胎兒蛋白的肝癌、肝硬化、與B型肝炎帶原者的病人），我們發現在Hp的N184位點上具有核心岩藻糖基與三岩藻糖基化三分支醣型結構在肝癌患者中顯著升高(含低濃度AFP之肝癌患者)。為了確定此結果，未來將持續增加樣本數來確認分析趨勢。

Disease biomarkers based on N-glycosylation profiles are explored actively because changes in the aberrations in glycosylation have been associated with several diseases, including cancer. Previously, our group has discovered potential HCC-elevated glycoforms through identification of glycosylation profiles of Haptoglobin (Hp) by untargeted mass spectrometry (MS) analysis and quantitative comparison across Hepatitis B virus (HBV), liver cirrhosis (LC) and Hepatocellular carcinoma (HCC) patients. The result revealed confident identification of 879 glycopeptides and 210 glycopeptides that show differential expression in HCC compared to HBV and LC. However, due to inferior sensitivity and limited duty cycle of untargeted MS methods, we switched to more robust targeted MS methods to verify our results. To confirm the potential utility of Haptoglobin (Hp) as a biomarker for hepatocellular carcinoma (HCC), in this work, we attempted to develop a targeted mass spectrometry approach to quantify site-specific glycosylation in selected glycoforms in Hp. Specifically, we developed a nanoprobe-based assay coupled to targeted MS methods, including multiple reaction monitoring (MRM-MS) and parallel reaction monitoring (PRM-MS) for multiplexed quantification of glycopeptides. Magnetic nanoprobes functionalized with hemoglobin (MNP@Hb) were used to purify haptoglobin (Hp) from serum. Following enzyme digestion, glycopeptides were enriched using hydrophilic interaction chromatography (HILIC). To ensure correct glycopeptides analysis, b and y ions from the peptide backbone were monitored, in addition to Y1 ion (peptide + HexNAc) and oxonium ions (HexNAc and HexHexNac). The oxonium ions were much more intense than the Y1, b and y ions, making oxonium ions suitable quantifiers, while the latter as qualifiers. On the method development using oxonium ions, the MRM-MS can get good linearity (r2 = 0.950~0.990) and sensitivity (62.5 ng Hp). However, its limited mass range posed a problem with detecting high molecular weight glycopeptides (m/z>1000). Thus, we took advantage of the wider mass range of PRM-MS, where we obtained good linearity (r2 = 0.996~0.997) and precision (CV=14.7%) for an Hp non-glycopeptide. We applied this method to quantify 104 Hp glycopeptides in liver diseases, 12 of which have good chromatographic trace and co-elution profile. Using clinical samples (n=5 each for high-HCC, low-HCC, LC and HBV), we found that a core-fucosylated form and tri-fucosylated tri-antennary form were significantly elevated in HCC, including low-AFP HCC. To confirm the result, analysis on a larger sample cohort is underway.