奈米探針親和質譜法檢測血清澱粉蛋白A異構物於不同疾病的表現差異

Expression Signature of Serum Amyloid A Variant for Detection in Different Diseases

陳肯(Michael Isaac Chen)

指導教授 ： 陳玉如

國立臺灣大學/理學院/化學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10685

摘要

疾病生物標記分子表現量的改變，常被應用於早期疾病診斷、檢示治療效果、監測疾病復發與手術預後評估。近年來，利用癌症病患血液中蛋白質表現量的顯著增加而應用於早期疾病診斷上逐漸受到注目。除了表現量的改變之外，基因變異與轉譯後修飾所導致的蛋白質變化，也被認為與疾病進程有相當的關連性。1,2先前已有報導指出，血清中的血清澱粉蛋白A(serum amyloid A, SAA)濃度與疾病，特別是癌症具有高度關聯性。然而，SAA 的同質異構體(protein variant)與癌症之間的關聯性到目前為止仍尚未有系統性的研究評估。
在本研究中，我們應用本實驗室所開發的奈米探針輔助親和性質譜法，藉由在磁性奈米粒子修飾具專一性的抗體，結合基質輔助雷射脫附/游離飛行時間質譜儀(MALDI-TOF MS)，對人體血清中的SAA同質異構體進行分離純化與偵測。藉由內標準品的添加，我們可以針對這些SAA同質異構體進行相對定量分析。本實驗中共分析了35例正常人、35例胃炎病患、70例胃癌病患、61例大腸癌病患與31例遺傳性多發腦梗死性癡呆病患。實驗結果顯示，我們總共偵測到24種不同的SAA同質異構體，包含SAA1與SAA2轉譯出來的蛋白質產物、多形態異構物(polymorphic isoforms)、N端胺基酸缺失的形態與另外三種尚未被報導過的SAA異構體。本論文更結合生物資訊方法，進一步建立了一套疾病鑑定的演算模組。在胃癌研究中，利用SAA同質異構體的表達形態，可以辨別胃癌病患與非胃癌個體，其靈敏度與專一性分別可達到0.76和0.81。在大腸癌研究中，SAA同質異構體的表達形態也可用來辨別大腸癌病患與正常人，其靈敏度與專一性則可達到0.69和0.82。然而，在遺傳性多發腦梗死性癡呆病患的分析上，SAA同質異構體的表達形態卻無法達到相同的疾病鑑別能力。此實驗結果暗示SAA同質異構體的生成機制與惡性腫瘤疾病的進展有相關性，與遺傳性基因變異疾病並無太大關連。

關鍵字

奈米探針親和質譜法 ； 血清澱粉蛋白A異構物 ； 生物標記分子 ； 胃癌 ； 大腸癌

並列摘要

Differential expression of disease-related biomarkers is the most common measure for early detection of disease, for monitoring effects of therapy, for detecting disease recurrence and for prognosis. Significant overexpression of plasma protein in cancer has received increasing attention in disease diagnosis. In addition to quantitative alterations in expression levels, genetic variations and post-translational modifications (PTMs) of proteins are also associated with disease states. Serum amyloid A (SAA), a major acute-phase protein, has received increasing attention due to its close association with inflammation. In previous reports, the expression of serum amyloid A (SAA) has been shown to be highly related to several diseases, especially cancer. However, the correlation of SAA variants with cancers has not been evaluated systematically.
Taking advantage of efficient affinity extraction by antibody-functionalized magnetic nanoparticles (MNPs) and accurate MALDI-TOF MS readout, we present a nanoprobe-based immunoassay for simultaneous isolation and screening of protein isoforms from human plasma. Relative quantification of these variants was performed by the addition of internal standard. Our results demonstrate that total 24 variants of SAA including SAA 1- and SAA 2- encoded protein products and their polymorphic isoforms, N-terminal truncated forms and three previously unidentified were identified from normal controls (n = 35), gastritis patients (n = 35), gastric cancer patients (n = 70), colon cancer patients (n = 61), and CADASIL patients (n = 31). Coupled with the bioinformatics study, we developed a computational model for cancer discrimination. In the gastric cancer study, the SAA variant pattern demonstrated discrimination of patients with gastric cancer from patients with gastritis or healthy subjects with sensitivity of 0.76 and specificity of 0.81. In the colon cancer study, the performance of SAA variant pattern has specificity of 0.69 and specificity of 0.82. However, the of SAA variants pattern did not show discrimination power in CADASIL study, which may indicate the SAA variants are highly related to malignant tumor-associated diseases.

並列關鍵字

nanoprobe-based affinity mass spectrometry (NBAMS) ； SAA variants ； biomarker ； MALDI-TOF MS ； gastric cancer ； colon cancer

參考文獻

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