開發凝集素奈米粒子結合親和質譜術之岩藻醣蛋白質體分析方法

Fucose-specific Glycosylation Profiling by Lectin-modified Nanoparticle and Mass Spectrometry

林怡茹(Yi-Ru Lin)

指導教授 ： 陳玉如

國立臺灣大學/理學院/化學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10569

摘要

中文摘要
岩藻醣基化(fucosylation)是岩藻醣以共價鍵結在其它單醣上的修飾，主要修飾在末端的單醣上稱為末端岩藻醣基化，或是最內側的乙醯葡萄糖胺(N-acetylglucosamine)，稱為核心岩藻醣基化。氮基醣鏈上不正常的岩藻醣基化常被應用於為許多癌症潛在的生物標記，例如像是前列腺癌、乳癌、肝癌、卵巢癌和胰臟癌等。一般來說，岩藻醣基化醣蛋白的鑑定主要可透過使用凝集素親和分析(lectin-affinity chromatography)結合質譜技術(mass spectrometry)來分析，但由於高度醣基異質性(heterogeneity)以及醣基化胜肽於質譜中的訊號易被未修飾胜肽抑制，使得岩藻醣基化醣蛋白在複雜樣品中難以被鑑定。
在本篇論文中，我們利用磁性奈米粒子容易被磁鐵分離和粒子上可以修飾的優點，提出以橙黃網胞盤菌凝集素(Aleuria aurantia lectin)鍵結於磁性奈米粒子(magnetic nanoparticle)的奈米探針技術作為岩藻醣基化醣胜肽純化方法，並且結合質譜技術為分析方法來鑑定醣胜肽。以山葵過氧化酶(horseradish peroxidase)作為岩藻醣基化醣蛋白的標準品，我們針對凝集素種類、磁性奈米粒子總量和溶液的極性和酸度等反應條件進行測試與優化岩藻醣基化醣胜肽純化方法。在優化條件下，以含20%乙醇的磷酸鹽緩衝液做為溶液，橙黃網胞盤菌凝集素修飾磁性奈米粒子(AAL@MNP)從山葵過氧化酶中純化11條岩藻醣基化醣胜肽，共含有已知的9個醣基化位置。於4種標準蛋白(山葵過氧化酶、胎球蛋白、核糖核酸酶和肌紅蛋白)的胜肽混合物中，山葵過氧化酶的岩藻醣基化醣胜肽也可被橙黃網胞盤菌凝集素修飾磁性奈米粒子純化，呈現出此奈米粒子對於山葵過氧化酶的岩藻醣基化醣胜肽的純化具有專一性(25%)。為了進一步了解瀰漫性大B細胞淋巴瘤(DLBCL)中醣蛋白質體學的性質，我們之後應用發展的方法純化並且鑑定瀰漫性大B細胞淋巴瘤其中的HBL-1 cell中蛋白質的岩藻醣基化。以氮基醣水解酶F(PNGase F)移除氮基醣鏈後，在HBL1細胞中分別以高密度和低密度的AAL@MNP純化，以Orbitrap Velos質譜鑑定到456條和433條去氮基醣鏈醣胜肽，分別來自於230個和229個HBL1細胞中的醣蛋白，而在完整氮基醣鏈醣胜肽，以MAGIC可以解出AAL@MNP純化後，9條醣胜肽中有8條為岩藻醣基化醣胜肽。其中包含了一些已知的岩藻醣基化醣蛋白，如CD44, IGHM, 和 GOLM1也可在此研究中鑑定出。我們也成功地利用此功能性奈米粒子純化並解析出分布在細胞表面的CD44帶有α1,2-岩藻醣鍵結之完整醣型，與存在B淋巴球表面受器的IGHM具有核心岩藻醣基化之結構。在分析完整的醣基化胜肽之醣結構結果中，利用oxinum ions鑑定到所有的醣胜肽圖譜中，HILIC有2743 張藻醣基化醣胜肽圖譜(專一性86.0%), AAL@MNP_LD 則有1419張 (84.0%), AAL@MNP_HD有1647張 (87.0%) 和LCA@MNP 有2616 張(74.9%)，表示利用岩藻醣專一凝集素結合奈米粒子技術對於岩藻醣基化醣胜肽的純化具有選擇性和專一性。綜合上面所述，橙黃網胞盤菌凝集素修飾磁性奈米粒子結合質譜技術提供一具有高度選擇性且專一性岩藻醣基化醣胜肽純化技術，並提升有效鑑定完整岩藻醣基化醣型與其修飾位置之分析，並且可應用於偵測癌症的潛在生物標記。

關鍵字

完整醣蛋白質體學 ； 岩藻醣基化醣胜肽 ； 橙黃網胞盤菌凝集素修飾磁性奈米粒子 ； B淋巴球 ； 質譜技術

並列摘要

Abstract
Fucosylation, covalent attachment of a fucose to other monosaccharides, is most commonly found to occur at the reducing terminal end (terminal fucosylation)　or at the inner core N-acetylglucosamine (GlcNAc) (core fucosylation). Altered fucosylation of N-linked glycans has been implicated and used as potential biomarkers in various cancers such as prostate, breast, liver, ovarian and pancreatic cancers. Analysis of fucosylated glycoproteins is usually accomplished by utilizing lectin-affinity chromatography combined with mass spectrometry (MS) identification. Due to high heterogeneity and signal suppression of the glycopeptides on MS analysis, analysis of fucosylated glycoproteins in complex samples remains challenges.
By taking advantage of the easy magnetic separation and flexible surface modification of the magnetic nanoparticle (MNPs), in this study, we proposed an integrated nanoprobe-based strategy using a fucose-specific Aleuria aurantia lectin immobilized magnetic nanoparticle (AAL@MNPs) for affinity enrichment of fucosylated glycopeptides (FPs) followed by MS analysis for glycopeptide (GPs) identification. Using horseradish peroxidase (HRP) as a fucosylated standard glycoprotein, we optimized the incubation conditions, including lectin type, MNPs amount, polarity and acidity of incubation buffer, for enrichment of fucosylated glycopeptides. Among the known 9 sites, total 11 FPs of 11 GP from HRP were enriched by AAL@MNPs with the optimal condition in 20% ACN with PBS (v/v). On the peptide mixture from 4 proteins (Horseradish peroxidase, Fetuin, Ribonuclease B, Myoglobin), AAL@MNP showed specificity (fucosylated peptides/the number of total glyco scan)(25%) on the enrichment of fucosylated glycopeptides on HRP. To understand the characteristics of fucosylation pattern in diffuse large B-cell lymphoma (DLBCL), we further applied the developed method for site-specific identification of fucosylated glycoproteome on activated B-cell-like DLBCL – and HBL-1 cells. With PNGase F deglycosylation, 456 and 433 de-N-glycopeptides corresponding to 230 and 229 glycoproteins in HBL1 cells were respectively enriched by high and low lectin density of AAL@MNP and identified by Orbitrap Velos MS. In intact glycopeptide part, our recently developed MAGIC was used to determine the glycan structure and peptide sequence; total 8 FPs from 9 glycoproteins were identified. The result identified some known fucosylated targets, such as IGHM, GOLM1 and CD44; CD44 has been reported to be a α1,2-fucose antigen-containing protein on cell surface and involved in the development, adhesion and metastasis of tumor cells. In the analysis of intact glycopeptides, we use the presence of oxinum ion to calculate the number of spectra for glycopeptide and fucosylated glycopeptide. About 2743 (specificity: fucosylated glycopeptide spectra/glycopeptide spectra=86.0%), 1419 (84.0%), 1647 (87.0%) and 2616 (74.9%) fucosylated glycopeptide spectra were identified from enrichment of HILIC, AAL@MNP_LD, AAL@MNP_HD, and LCA@MNP, respectively. It indicated that our AAL@MNP strategy presented selectivity and specificity for fucosylated glycopeptides. In summary, the AAL@MNP-based mass spectrometry method demonstrated fucose-specific enrichment identification of fucosylated glycoproteome, facilitating the applications on the discovery of new potential diagnostic and therapeutic markers.

並列關鍵字

intact glycoproteome ； fucosylation ； Aleuria aurantia lectin ； magnetic nanoparticle ； B lymphoma cell ； mass spectrometry.

參考文獻

Reference

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