以奈米探針親和質譜法分析單一醣蛋白質體學於肝疾病中之差異

Abstract

現今診斷肝癌最常見的方法，如超音波掃描影像檢查或血清檢驗甲型胎兒蛋白(alpha-fetoprotein, AFP)濃度，都仍無法正確分辨癌症腫瘤特性或對甲型胎兒蛋白偵測的靈敏度及特異度不足。為了找尋出高靈敏度以及特異性的理想腫瘤標記分子，許多研究開始朝向分析肝疾病中標記蛋白的醣基化修飾與其變化。由許多文獻指出，血紅素結合蛋白(Haptoglobin, Hp)上的醣基化修飾的變化跟發展成肝癌的過程中有很大的關係，因此在本篇論文中，我們發展出兩種不同的親合性奈米探針結合質譜分析技術，對於單一生物標記血紅素結合蛋白上醣基化修飾在肝疾病中的改變研究，以找出早期診斷肝癌的腫瘤標記分子。 第一個策略是用磁性奈米粒子修飾上跟目標蛋白血紅素結合蛋白有親和性的血紅蛋白(Hemoglobin, Hb)，純化出血液中的血紅素結合蛋白，再利用親水性作用層析法(Hydrophilic interaction chromatography，HILIC)萃取其醣胜肽，並結合質譜分析以鑑定特定位點之醣型。此研究分析了33例肝疾病病患，包括11例肝癌、11例肝硬化以及11例B型肝炎病人。我們成功地在肝癌、肝硬化以及B型肝炎病人分別鑑定到183、169、164條醣胜肽，醣基化的位置是位於天冬醯胺(Asparagine, Asn) Asn184, Asn207, Asn211以及Asn241。由比對醣胜肽的結果得知，約有2種雙角分支之唾液酸醣結構可在每一肝疾病族群中大於10個病人以上鑑定而得。此外，我們鑑定到28個獨特的醣胜肽在肝癌中出現，其中包括了5個雙角分支及三角分支的核心岩藻醣型，推測這些特定位點的獨特醣型可能有助於區分肝癌、肝硬化與B型肝炎病人。因此，這些常見或獨特的岩藻醣基化或是帶有不同分支的醣結構在不同醣基化位置的變化，將可提供肝癌在早期診斷的新方向。 在第二部分，我們發展出一鍋化(One Pot)策略對於蛋白質以及醣胜肽純化以及生物標記分子醣基化修飾的鑑定。同時利用兩種奈米探針：修飾上血紅蛋白的二氧化矽奈米粒子(Hb@SiO2)純化目標蛋白; 修飾上配位體的磁性奈米粒子(ligand@MNP)純化出醣胜肽。經過條件最佳化以及方法評估後，此一鍋化方法可以減少兩倍以上的醣胜肽含量和醣胜肽鑑定數目的流失。我們將此策略也應用在肝病病患的分析上(三例肝癌，三例肝硬化及三例B型肝炎病人)，亦有效鑑定到雙角分支之岩藻醣基化結構只會在肝癌中出現，且不存在於肝硬化病人以及B型肝炎病人中。此一鍋化方法不僅可以增加醣胜肽的鑑定，對於特定醣結構(岩藻醣基化以及唾液酸苷化)的研究也有更好的偵測靈敏度。

Current diagnostic approaches of Hepatocellular Carcinoma (HCC) such as ultrasonography and serum alpha-fetoprotein lack satisfactory sensitivity and specificity. To address this issue, researches have shifted towards analysis of altered glycosylation patterns of proteins in liver diseases. Toward biomarker discovery, in this thesis, we aim to analyze the glycosylation profiling of haptoglobin (Hp), a well-known acute phase protein associated with liver cancer progression, among liver diseases. On the technical front, we present two single glycoprotein-omics strategies to comprehensive intact glycosylation analysis of biomarker candidates. We expect to identify the unique and aberrant glycoforms at specific N-glycosylation sites of Hp, which may serve as biomarker candidate for early diagnosis of liver diseases. The first strategy uses antibody-conjugated magnetic nanoprobes for Hp enrichment from human serum, followed by hydrophilic interaction liquid chromatography for glycopeptide enrichment and identification by mass spectrometry (NP-HILIC-MS). This strategy was employed for Hp profiling of serum from 33 individual patients: 11 HCC patients, 11 liver cirrhosis patients (LC) and 11 Hepatitis B (HBV) inactive carriers. We achieved site-specific glycosylation profiling for total of 183, 169 and 164 intact glycopeptides in HCC, LC, and HBV serum samples, respectively from Asn184, Asn207, Asn211 and Asn241 sites. Two common sialylayted bi-antennary glycopeptides were identified in 10 of 11 patients for each cohort. More importantly, 28 unique glycopeptides containing 5 core fucosylated bi- and tri-antennary structures, were uniquely discovered in HCC, suggesting that the site-specific unique glycoforms might be used to discriminate HCC from LC and HBV patients. With verification of larger cohort, the glyco-signature at specific glycosylation sites on Hp will provide a new opportunity for early diagnosis of liver diseases. In the second part, we developed a novel method to improve sensitivity for detecting specific glycopeptides: we developed a One Pot enrichment strategy for concomitant purification and glycosylation profiling of biomarker candidates in serum. This approach takes advantages of dual nanoprobes: hemoglobin-conjugated silica nanoparticle (Hb@SiO2) for enrichment of target protein and ligand-conjugated magnetic nanoparticle (ligand@MNP) for enrichment of target glycopeptide. After optimization, this one pot method enhanced the number (2-fold) and abundance (2-fold) of glycopeptides. On the application to liver disease patients (HCC, n=3; LC, n=3 and HBV, n=3), unique bi-antennary fucosylated glycopeptides were found in HCC patients, but not in LC and HBV patients. We conclude that One Pot method can be utilized to target specific glycoforms (fucosylation and sialylation) in liver diseases and enhance detection sensitivity.