優化血漿蛋白質體學應用於可逆性腦血管收縮症候群之分析;Development of an Optimized Plasma Proteomics Workflow and its Application in Reversible Cerebral Vasoconstriction Syndrome (RCVS)

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题名:	優化血漿蛋白質體學應用於可逆性腦血管收縮症候群之分析;Development of an Optimized Plasma Proteomics Workflow and its Application in Reversible Cerebral Vasoconstriction Syndrome (RCVS)
作者:	莊怡煊;Chuang, Yi-Shuang
贡献者:	化學學系
关键词:	血漿蛋白質體學;可逆性腦血管收縮症候群;非數據依賴擷取方法
日期:	2025-08-18
上传时间:	2025-10-17 11:36:25 (UTC+8)
出版者:	國立中央大學
摘要:	基於質譜的血漿蛋白質體學研究中，蛋白質覆蓋率低，是由於血液中蛋白質動態範圍大（>10數量級)，高含量蛋白質的信號過強導致低含量蛋白質不易被偵測到，因此開發出了去除策略，將高含量蛋白質，例如白蛋白、纖維蛋白、IgA、IgG等常出現於血漿中的蛋白經由免疫耗盡法去除，降低高含量蛋白質的訊號以利低含量蛋白質被偵測，增加蛋白質覆蓋率。可逆性腦血管收縮症候群(RCVS)是一種中樞神經疾病，在發病期中會出現雷擊式頭痛，而目前針對RCVS尚無特效藥，且對於發病機轉尚不清楚，在一般醫院的檢測是透過斷層掃描及核磁共振來判定。在先前的研究中，使用了RCVS患者的腦脊髓液作為分析樣品，利用蛋白質體學方法嘗試找出與RCVS相關的生物標記分子，並假設相關訊號途徑。本篇研究首先優化了血漿蛋白質體學研究流程，在免疫耗盡法中做試劑的選擇，為了因應高通量樣品做了除鹽步驟的優化，且在質譜參數上也針對掃描範圍(scan range)、解析度(orbitrap resolution)以及隔離寬度(isolation window)做了最佳化調整。接著分析健康人、RCVS病患在急性期與恢復期三群體之間蛋白的表現，經由變異數分析(ANOVA)結果表示，顯著差異蛋白大部分與補體、發炎、凝血以及免疫相關，而在先前文獻中提到的12個候選生物標記，在血漿樣品中有6個被偵測到，分別為FCGR3A、PRNP、ANXA1、ART3、CANX、THBS1。經由兩兩T-test 檢定，其中ART3與PRNP在健康人與RCVS急性期患者、健康人與RCVS恢復性期患者中有顯著差異。PRNP在健康人與急性患者有相同的分佈趨勢，在健康人中較低，在急性患者中較高；但在ART3中，健康人與急性患者有相反的分佈趨勢，CSF樣品在健康人中較低，在急性患者中較高，血漿患者則是相反，在健康人中較高，在急性患者中較低。此發現改善可逆性腦血管收縮症候群使用腦脊髓液作為樣品不易取得問題，亦能夠了解先前研究在腦脊髓液中作為生物標記的蛋白在血漿中的分佈情形，對於深入發展可逆性腦血管收縮症候群生物標記以及了解疾病狀態具有優勢，期許能夠對於醫療診斷提供更多的資訊。;In mass spectrometry-based plasma proteomics, low protein coverage remains a major challenge due to the wide dynamic range of plasma proteins (>10 orders of magnitude). Signals from high-abundance proteins such as albumin, fibrinogen, IgA, and IgG often overshadow low-abundance proteins, making their detection difficult. To overcome this, depletion strategies have been developed to remove high-abundance proteins through immunoaffinity methods, thereby enhancing the detection of low-abundance proteins and improving proteome coverage. Reversible cerebral vasoconstriction syndrome (RCVS) is a central nervous system disorder characterized by sudden, thunderclap headaches during the acute phase. Currently, there are no specific treatments for RCVS, and its underlying mechanisms remain poorly understood. Diagnosis in clinical settings typically relies on CT or MRI imaging. In previous studies, cerebrospinal fluid (CSF) samples from RCVS patients were analyzed by proteomics to identify potential disease-associated biomarkers and implicated signaling pathways. In this study, we first optimized a plasma proteomics workflow. This included evaluating different immunodepletion reagents, streamlining desalting methods for high-throughput processing, and fine-tuning LC-MS/MS parameters such as scan range, Orbitrap resolution, and isolation window. Using the optimized workflow, we analyzed protein expression across three groups: healthy individuals, RCVS patients during the acute phase, and the convalescent phase. ANOVA revealed a total of 258 differentially expressed proteins, many of which are related to complement activation, inflammation, coagulation, and immune response. Furthermore, of the 12 previously reported CSF-based candidate biomarkers, 6 were successfully detected in plasma: FCGR3A, PRNP, ANXA1, ART3, CANX, and THBS1. Among these, PRNP and ART3 showed significant differences between healthy controls and both acute and convalescent RCVS patients based on pairwise t-tests. Interestingly, PRNP exhibited consistent trends between CSF and plasma samples, with elevated levels in RCVS patients compared to controls. In contrast, ART3 displayed opposite expression patterns: elevated in CSF but decreased in plasma during the acute phase, suggesting compartment-specific regulation. These findings highlight the feasibility of using plasma as an alternative to CSF for biomarker discovery in RCVS, a clinically relevant advancement given the invasiveness of lumbar puncture. Moreover, this work contributes to our understanding of how CSF-derived biomarkers behave in circulation and lays the groundwork for developing accessible, blood-based diagnostic tools for RCVS.
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