以二甲基標記結合質譜技術進行芒果過敏原之定性與定量分析

Abstract

食物過敏是重要的食安議題之一，建立一套可靠的過敏原定量方法，在食品安全管理上是必需的。因此，開發一套穩定、準確的過敏原分析方法是非常重要的。以質譜法作為過敏原檢測之方式，具有高穩定度、高準確度等優點，故近年來逐漸受到重視。芒果 (Mangifera indica) 為我國常見的水果，且其亦為我國主要致敏性水果之一，但目前研究對芒果過敏原身分及蛋白質序列了解較少，本研究選擇以愛文芒果為目標，以質譜儀進行過敏原身份鑑定與定量分析。結果分為三部分：第一部分，為取得愛文芒果過敏原的完整蛋白質序列以設計代表胜肽，首先建立愛文芒果轉錄體資料庫。實驗上以次世代定序方式進行愛文芒果轉錄體定序，進行組裝後得到 87,245 條 contigs，經註解得 47,921 個 unigene，再將基因序列轉譯後，即可得到愛文芒果的蛋白質序列資料庫，作為過敏原身份鑑定比對之用。第二部分，進行愛文芒果高致敏性蛋白質身份鑑定。切下 SDS-PAGE 上分子量接近 30 kDa 與 67 kDa 位置之蛋白質進行膠內消化及質譜分析，並將分析結果與第一部分建立之資料庫進行比對以確認其身份，結果顯示愛文芒果之 30 kDa 過敏原 Man i 2 很可能為 chitinase，而 67 kDa 過敏原則很可能為 heat shock protein 70 kDa (hsp70)。第三部分，建立愛文芒果過敏原質譜定量平台。從質譜分析結果與前人研究中選擇chitinase、GAPDH 與 hsp70 代表胜肽，以二甲基標記製備內標準品與外標準品，並建立定量平台，氫標記之 chitinase、GAPDH 及 hsp70 代表胜肽 (chitinase_H、GAPDH_H 及 hsp70_H) 定量極限分別為 4.5 ng/mL、22.5 ng/mL 及 0.225 ng/mL，而 chitinase_H 及 hsp70_H 三個確效濃度之回收率分別為 90.4%-91.6%及 88.7%-97.6%，將此法用於定量愛文芒果鮮果樣品中過敏原含量，結果顯示三種過敏原於愛文芒果中之濃度皆低於本方法之偵測極限。本研究成功建立愛文芒果轉錄體資料庫，及成功對 2 個芒果中高致敏性蛋白質進行身份鑑定，該轉錄體資料庫可供未來愛文芒果蛋白質體學相關研究使用。同時建立以質譜儀為基礎之芒果過敏原定量法。本研究可作為未來食品過敏原方法開發之參考，並應用於各國以提升食品安全。

Food allergy is an important food safety issues in the world, therefore, a reliable allergen quantification method is required to enforce the food control. With high precision and accuracy, mass spectrometry-based method have been applied for allergen detection. Mango (Mangifera indica) is one of the major allergenic food in Taiwan. Nevertheless, research on identity and protein sequence of mango allergens is limited. In this study, we choose Irwin mango as a target, and identification of the allergens and the establishment of MS-based allergen quantification platform were carried out. Our results consists of three parts. In the first part, establishment of transcriptome database of Irwin mango was performed. In order to obtain the full sequence of Irwin mango and design signature peptides, transcriptome database of Irwin mango was established by next generation sequencing. After assembly process, there were 87,245 contigs obtained, which were annotated to 47,921 unigenes. The nucleotide sequences of unigenes were translated to amino acid sequences as a reference for subsequent protein identification. In the second part, the identification of two mango allergens was performed. Proteins located at 30 kDa and 67 kDa on SDS-PAGE were subjected to in-gel digestion and mass spectrometry (MS) analysis. The results showed that the 30 kDa and 67kDa allergen of Irwin mango were identified as chitinase and heat shock protein 70 kDa (hsp70). In the third part, a MS- based quantification method for Irwin mango allergen was established. Signature peptides of chitinase and hsp70 were selected from results of MS analysis, and signature peptide of GAPDH was determined according to previous study. Dimethyl labeling was used to produce internal and external standard, and the H-labeled standards were named as chitinase_H, GAPDH_H and hsp70_H. Limit of quantification of chitinase_H, GAPDH_H and hsp70_H was 4.5 ng/mL, 22.5 ng/mL and 0.225 ng/mL, respectively. Recovery of chitinase_H and hsp70_H was 90.4%-91.6% and 88.7%-97.6%. Finally, the established method was applied to mango fruit, and the results showed that the concentration of these allergens was below limit of detection. In conclusion, we established the transcriptome database of Irwin mango and identified 2 major allergens of Irwin mango. Besides, the transcriptome database could be used for future research on mango proteomic. We also developed a MS-based method for the quantification of mango allergen. Our study could not only be an example of analytical method development, but also could be use in the world to improve food safety.