利用穩定同位素二甲基標記結合質譜技術檢測番茄過敏原 Sola l 1 之研究

Abstract

番茄為台灣一重要的致敏蔬果，近年來 Sola l 1被歸類為番茄果實中一個新的次要過敏原，因其序列保守且結構相近而時常造成食物與花粉間的交叉反應導致過敏現象。免疫分析法為目前最常用於檢測食品過敏原的方法，然因交叉反應的存在而導致其可能產生錯誤的結果而無法有效偵測過敏原，因此，須尋求特異性更加的番茄過敏原分析方法。質譜技術具有高特異性、高靈敏度、高準確度及分析速度快等特點，而常被用於各領域之定性與定量分析，其中 MRM 為目前串聯質譜中最常用於定量的方式。穩定同位素二甲基標記因具有高效、快速、簡便、廉價等優點，近年來已被廣泛用於蛋白質體學上的定量，然而目前卻無人將其應用於植物性蛋白上。故本研究目的在於利用穩定同位素二甲基標記結合質譜技術開發新方法，並以此建立番茄過敏原 Sola l 1 的定量平台，進而應用於食品分析之領域。 研究主要分為三個部分：第一部分為利用重組蛋白 rSola l 1 搭配 Q-TOF 進行定性分析，並找出一段穩定且適合的胜肽序列作為目標蛋白之標準胜肽，結果表明，利用 trypsin 進行水解可得到一段帶有 13 個胺基酸之獨特且可信的胜肽片段 YR，確認後將該序列合成以進行後續分析；第二部分藉由甲醛氫同位素與氘同位素試劑分別對 YR 進行標定，其結果顯示兩者標記後皆無殘留之原胜肽，說明其具有良好的標記效率；而後將 YR-H 配製成不同濃度，並加入固定量之 YR-D 作為內標準品以 QqQ 繪製檢量線，進而對番茄中之 Sola l 1 蛋白進行定量，該檢量線表明胜肽濃度範圍為 0.5-2500 ng/mL 時與質譜反應量之間具有良好的線性關係；第三部分為探討該分析方法並予以確效，藉由線性、回收率、偵測極限及定量極限、重複性等評估此分析方法是否適用，結果發現，該方法皆適用於番茄重組蛋白和標準胜肽，且具有良好的線性和準確度，搭配 MRM 所得之重組蛋白偵測與定量極限皆為 0.5 μg/mL，標準胜肽樣品之偵測與定量極限最低則分別可至 0.1 ng/mL 和 0.5 ng/mL，展現其於蛋白質定量上之潛力。

In Taiwan, tomato is one of the most important vegetables that frequent causes of allergy. Recently, Sola l 1 was confirmed as a new minor allergen in tomato fruits. Because of the highly conserved sequence of protein structure among profilins, highly cross-reactive usually happen between pollen and foods and then caused allergic symptoms. Immunization assay is the most common method to detect food allergens. However, it may lead to false positive or false negative results due to the presence of cross-reactions. Therefore, we must to seek a specific method to analyze tomato allergens. Mass spectrometry has the advantage of high specificity, high sensitivity, high accuracy and rapid analysis, so it is used in many different fields for qualitative and quantitative analysis. Multiple reaction monitoring (MRM) is the most common method for quantitation with tandem mass spectrometry. Stable isotope dimethyl labeling has been widely used in quantitative proteomics due to it is highly efficient, fast, simple and inexpensive, but nobody applies it to vegetable protein sample. The purpose of this study was to develop a novel strategy by using stable isotope dimethyl labeling with LC - MS/MS, and establish the analysis platform for tomato allergen Sola l 1 then applied to the fields of food analysis. First, we analyzed the recombinant protein rSola l 1 by trypsin digestion and identified its sequence by Q-TOF, then found a suitable peptide as the standard peptide of target protein. The qualitative result showed that the peptide YR, which has 13 amino acids in length, was stable and unique. The standard peptide was synthesized and used to synthesized for further experiments. Second, the synthetic peptide YR was labeled with formaldehyde-H2 and formaldehyde-D2, respectively. The dimethylated tryptic peptides were subsequently analyzed by Q-TOF. The result indicated the YR peptides obtain a complete labeling of N-terminal residue, and it represented that the method equipped with a good labeling efficiency. A series of different concentration of light stable isotope dimethyl-labeled synthetic peptides (YR-H) were prepared, and fixed concentration of intermediate stable isotope dimethyl-labeled synthetic peptides (YR-D) were spiked as internal standards. The calibration curve was obtained and indicated a good linear relationship of the concentration of YR-H between 0.5 ng/mL to 2500 ng/mL, and it could be used for quantitation of target protein. Further, we validated the method through evaluating its linearity, recovery, limit of detection and quantification and repeatability, the result showed that dimethyl-labeled could be applied to recombinant protein and standard peptides, and it was reliable and accurate. The detection and quantification limits (LOD and LOQ) that analyzed by MRM of recombinant protein were both 0.5 μg/mL, and standard peptides were 0.1 ng/mL and 0.5 ng/mL, respectively, and this method showed potential for comprehensive quantification of protein.