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| 題名: | 開發液相層析-串聯式質譜儀方法相對定量 IgG、IgA、和 IgM 及其醣化修飾:應用於肺結核感染生物標記探勘
Development of UHPLC-MS/MS Method for Relative Quantification of IgG, IgA, and IgM and The Glycopeptides: Application to The Discovery of Biosignatures for Tuberculosis Infection |
| 作者: | 鄭玉暄
CHENG, YU-HSUAN |
| 貢獻者: | 醫學科學研究所碩士班
蔡伊琳 |
| 關鍵詞: | 免疫球蛋白M;N醣基化;結核病;液相層析質譜儀
IgM;N-glycosylation;tuberculosis;LC-MS/MS |
| 日期: | 2021-07-07 |
| 上傳時間: | 2022-03-23 22:48:50 (UTC+8) |
| 摘要: | 結核病是全球前十大死因之一，根據臨床症狀與傳播方式可分為潛伏性結核病(LBTI)和活動性結核病(ATB)，因為潛伏性結核病的診斷較為困難且不易察覺疾病狀態的變化，5-10%未治療的潛伏性結核病患者容易惡化為活動性結核病。本篇研究中，我們首先利用液相層析-串聯式質譜儀聚焦於IgM N-醣基圖譜的偵測方法開發。藉由特殊的Lambda和Kappa light chain親和珠可在純化的過程中將IgG、IgA與IgM同時純化，因此可以同時分析IgG、IgA、IgM及其醣基圖譜。我們優化樣品前處理的條件，並且加入與醣胜肽(glycopeptide)序列相似的同位素標記胜肽當作內標以建立醣胜肽的相對定量方法。IgG、IgA、和IgM檢量線的相關係數高於 0.989，而醣胜肽的回歸線(線性/二次回歸)相關係數則是高於 0.979。除此之外，我們也分析了相對定量方法的重複性(repeatability)，在同批 (intra-batch)與不同批(inter-batch)樣品間，精確度相對標準差的範圍分別落在1.40% 至 29.64% 和2.32% 至26.11%。最後，我們將所開發方法應用於臨床檢體上，以監測結核病感染的疾病狀態與醣基圖譜的關係。我們發現在結核病患者中，fucosylated 、sialylated和galactosylated的抗體比健康人還多，並且我們篩選出8個具有潛力的Fc glycopeptides作為區分活動性與潛伏性結核病的生物標記。我們期待藉由本篇研究，可以使免疫球蛋白IgG、IgA、和IgM醣基化的分析平台更加完備，並且開發的醣基與定量方法除了應用於肺結核之外，可能還可以應用於其他感染性疾病上。
Tuberculosis (TB) is one of the top 10 causes of death worldwide. According to the symptoms and transmissibility, TB infection can be divided into latent TB infection (LTBI) and active TB infection (ATB). This study developed an LC-MS/MS method to investigate human plasma IgM and its N-glycosylation features. By using lambda and kappa light chain affinity matrixes, we could purify IgM as well as IgG and IgA from human plasma simultaneously. Parameters in terms of bead volumes, incubation times, and enzymatic digestion time were optimized. Stable isotope labeled peptides that were similar to the glycopeptides were used to compensate for the variation during enzymatic digestion and UHPLC-MS/MS analysis. We also established a relative quantitation workflow for 136 glycopeptides of IgG, IgA, and IgM. The correlation coefficients for all glycopeptides were higher than 0.979, and the ones for IgG, IgA, and IgM surrogate peptides were higher than 0.989. For repeatability analysis of the developed method, the intra-batch precision of targeted glycopeptides was ranged from 1.40% to 29.64% RSD, and the inter-batch precision was ranged from 2.32% to 26.11% RSD. Finally, we applied the developed method to clinical plasma samples from patients infected with tuberculosis and healthy controls to monitor their disease status and glycosylation patterns. We found that the abundance of fucosylated, sialylated and galactosylated antibodies were higher in patients with tuberculosis infection. We also selected 8 Fc glycopeptides as potential biosignatures for ATB and LTBI discrimination. The findings in this study provided the fundamental understanding of site-specific glycosylation patters in tuberculosis for IgG, IgA, and IgM, and the developed method could be potentially applied to other infectious disease as pilot studies. |
| 描述: | 碩士
指導教授：蔡伊琳
委員：王三源
委員：張心儀 |
| 資料類型: | thesis |
| 顯示於類別: | [醫學科學研究所] 博碩士論文
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