優化高含量蛋白去除策略用於全面性分析人類血液蛋白體

An optimized high abundant protein depletion strategy for large-scale profiling of human blood proteome

陳宜虹

血清 ； 血漿 ； 蛋白移除 ； 蛋白質體 ； Serum ； Plasma ； Protein depletion ； Proteomics

臺北醫學大學臨床藥物基因體學暨蛋白質體學碩士學位學程學位論文

2017年

碩士

韓嘉莉

繁體中文

在各式各樣的臨床檢體中，血漿和血清是容易取得且適用於開發監測疾病進程或治療療效之生物標記候選蛋白質的樣品。然而，血液內的蛋白質濃度差異大、組成複雜，使得偵測具有臨床重要性的低含量蛋白變得十分困難。為了克服這項瓶頸，我們提出著重於去除血漿及血清內高含量蛋白的樣品處理優化策略。具體來說，本篇研究評估三種以抗體為基礎的去除方法，包含Aurum血清蛋白移除管柱、ProteoPrep20 血漿蛋白移除管柱和MARS多親和性蛋白移除管柱等，以及三種無抗體的沈澱去除方法（乙晴、三氯乙酸/丙酮和聚乙烯二醇）。我們以川崎氏症病人血漿及9位纖維肌痛症病人的混合血清作為評估方法的標準樣品。 在以抗體為基礎的單一去除方法中，MARS蛋白移除管柱相比於ProteoPrep20（80%）與Aurum（61.4%），可以達到較高的高含量蛋白移除量（約95%），且鑑定較多的胜肽及蛋白數量。在無抗體的單一沈澱去除方法中，三氯乙酸/丙酮沉澱能移除68%的蛋白量，相比於純血清可增加1.16倍胜肽與1.14倍蛋白鑑定數，乙晴沉澱法則是出現嚴重的樣品損失，導致鑑定的蛋白數量降為純血清的0.68倍，聚乙烯二醇沈澱因需要進行多種濃度之序列沈澱而花費較多時間，也可能導致定量誤差。基於單一移除方法的結果，我們選擇MARS和三氯乙酸/丙酮沈澱形成串聯去除法以移除血清中的高含量蛋白。結果顯示先使用MARS再使用三氯乙酸/丙酮沉澱的串聯去除法顯著降低前14名高量蛋白的血清含量（從71.2%降至0.6%），且相比於純血清可增加胜肽（2.18倍）和蛋白（1.24倍）的鑑定數量。 我們將此優化的串聯去除策略應用於進行纖維肌痛症病人及健康人的血清蛋白體定量分析，結果發現在病人血清中，FN1、APOA4及C1QA等三個蛋白含量升高，而C3及APOH的含量卻是下降。在這些蛋白中，FN1在病人血清有12.12倍的高表現量，其為細胞外基質骨架的組成成分，且已知參與調控腎小球病變伴隨fibronectin沉積疾病。透過完成本篇研究，我們期望此優化之串聯去除法可成為全面性分析血液蛋白體的有效樣品處理策略。

Among various clinical specimens, plasma and serum is an easily accessible sample for discovery of biomarker candidate proteins associated with disease progression or treatment response. However, the wide dynamic range and complex compositions in plasma/serum present great challenges to identify low abundant proteins of clinical importance. To overcome these bottlenecks, we proposed an optimized sample preparation strategy, which emphasized on the depletion of high abundant proteins in plasma and serum. Specifically, three kinds of antibody-based depletion kits including Aurum serum protein mini kit, ProteoPrepR 20 plasma immunodepletion kit, and Multiple affinity removal spin cartridge human-14, and three antibody-free precipitation methods (ACN, TCA/acetone, and PEG) were evaluated in this study. Plasma samples from kawasaki disease patients and pooled serum samples from 9 fibromyalgia patients were used as references for comparison. For single depletion by antibody-based kits, the MARS kit achieved higher (~95%) removal of high abundant proteins compared to ProteoPrep20 (80%) and Aurum (61.4%) and facilitated more identified peptides and proteins. For single depletion by antibody-free methods, TCA/acetone precipitation could remove 68% of protein amount and increase the 1.16-fold number of identified peptides and 1.14-fold proteins compared to raw serum. A significant sample loss was observed by using ACN precipitation with lower identified proteins (0.68-fold) compared to raw serum. Longer preparation time was required for sequential precipitations by multiple PEG concentration which may lead to higher quantitation variation. Based on the single depletion results, we selected MARS and TCA/acetone precipitation as tandem depletion to sequentially deplete high abundant proteins in serum. The results showed that tandem depletion which integrated first MARS depletion and second TCA/acetone precipitation would significantly decrease (from 71.2% to 0.6%) the abundances of top 14 high abundant proteins and increase the number of identified peptides (2.18-fold) and proteins (1.24-fold) compared to raw serum. The optimized tandem depletion strategy was applied to quantitative analysis of proteome profiles in serum samples from fibromyalgia patients and healthy controls. Three proteins, FN1, APOA4 and C1QA, were identified with elevated expressions and C3 and APOH were lower expressed in fibromyalgia patients. Among these proteins, FN1, which is an extracellular matrix structural constituent and have been reported to involve in glomerulopathy with fibronectin deposits 2 disease, showed 12.12-fold higher expression in fibromyalgia patients. Through the completion of this study, we expect that our optimized tandem depletion would be an efficient workflow for large-scale quantitative profiling of blood proteome.

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