利用二甲基標記結合質譜技術建立奇異果過敏原之絕對定量分析平台

Abstract

食品過敏為重要之食品安全議題，而過敏原之定量法為其管理上所必需的。市面上最普及的ELISA法，方法立基於抗體，亦受抗體特性所限制，具有許多難以改善之缺點。近年來以質譜為基礎之過敏原定量法逐漸受到重視，因其不僅能解決ELISA法之缺點，同時具有高通量、能提供分子指紋證據等優點。在食品過敏原中以水果過敏原之定量，因為蛋白質含量低、干擾物質多故較為困難。奇異果 (Actinidia deliciosa)為國人大量食用之水果，亦為致敏性食物，於歐洲食物過敏原排名前六，於台灣排名前十，為我國建議標示之食品過敏原。本研究選擇奇異果作為代表物進行質譜過敏原定量法之開發。 結果分為三部分：第一部分、以奇異果為例之水果過敏原質譜前處理法開發與優化。開發出兩種適合水果之苯酚蛋白質萃取法，分別為最佳化苯酚蛋白質萃取法 (optimized phenol extraction method, OPE) 及微量苯酚萃取法 (micro phenol extraction method, MPE)，兩者皆具高萃取率。其中OPE單次萃取蛋白質總重量較MPE多；而MPE之成本低、操作簡便快速 (1天)、具良好之消化步驟相容性。同時優化胰蛋白酶水解法與確認二甲基穩定同位素標記法之標記效率與同位素效應，並將MPE、胰蛋白酶水解法、二甲基標記法與樣品除鹽法，整合為一套可連續且低轉換損耗之質譜樣品前處理法。第二部分、建立及確效奇異果過敏原之質譜絕對定量分析法。利用quadrupole time of flight mass spectrometer (Q-ToF MS) 結果來進行奇異果目標過敏原之選擇。以Act d 1、Act d 5、Act d 11作為目標物，AD1、AD5、AD11胜肽分別作為其代表胜肽。再以triple quadrupole mass spectrometer (QqQ MS)選擇代表胜肽之定量子離子與定性子離子及進行LC與MS之參數調整。對本法進行確效試驗，AD1H、AD5H胜肽之定量範圍分別為1250-125000 ng/mL、2500-250000 ng/mL；準確度(回收率)分別為82.76%、83.46%；重複性(變異係數)分別為6.61%、8.69%；儀器定量極限分別為0.008 ng/mL、8 ng/mL；儀器偵測極限分別為0.008 ng/mL、4 ng/mL；兩者皆具良好專一性及線性，成功建立於多重反應監測模式下之奇異果過敏原質譜分析法。第三部分、將此法應用於奇異果加工製品中。本研究比較了鮮果、果醬、熱風乾燥果乾、冷凍乾燥果乾、熱殺菌果泥、高靜水壓果泥、熱殺菌果汁與高靜水壓果汁，八種常見之奇異果產品於加工後過敏原之消長。此方法成功定量出八個處理組之Act d 1、Act d 5含量，證明了本法適用於上述加工奇異果加工製品中。於長時間加熱之加工製品中 (果醬與熱風乾燥果乾) 過敏原含量較低，而熱殺菌、高靜水壓或冷凍乾燥製程對於過敏原含量較無影響。 本研究成功開發出OPE與MPE兩種水果蛋白質萃取法，並將MPE、蛋白水解法、標記法與樣品除鹽法整合優化為一套質譜樣品前處理法，及成功建立奇異果過敏原質譜絕對定量分析法，並成功將此方法應用於八種奇異果食品中。本研究可作為未來食品過敏原方法開發之範本，並於未來能應用於各國，協助食品安全之提升。

Food allergy management is one of the main issues in food safety. For efficient management of allergy, an effective analytical method is required. In the past, antibody-based Immuno-assay ELISA was the most popular analytical method for allergen determination, however it has some disadvantages in the use of antibody. Recently, mass spectrometry-based methods gains popularity because it can overcome the disadvantages of antibody-based methods, also possesses advantages such as high throughput, and ability to provide molecular evident etc. Quantification of plant allergens, especially fruit allergens, which have low protein contents, is difficult to achieve, compared to other food allergens. Kiwi fruit (Actinidia deliciosa) is one of the popular fruits in Taiwan. It ranks the second highest among the imported fresh fruits. Kiwi fruit is also an allergenic fruit, of which allergic popularity is rated sixth among food allergens in Europe and tenth in Taiwan. Many governments suggest allergenic labeling on food label. In our study, we choose kiwi fruit as an example to develop a MS-based quantification method for fruit allergen. Our results consisted of three major parts. In the first part, we have developed and optimized sample preparation methods for mass spectrometry analysis of fruit allergen using kiwifruit as a model sample. We have developed two fruit optimized phenol extraction methods, including optimized phenol extraction method (OPE) and micro phenol extraction method (MPE). Both methods had yielded a high extraction efficiency. OPE had a higher protein extraction weight, while MPE had some other advantages such as low cost, fast, simple and was well compatible with further mass spectrometry sample preparation steps. Meanwhile we have optimized trypsin in-solution digestion method and confirmed efficiency as well as isotopic effect of stable isotope dimethyl labeling method. We have also combined different methods of sample preparation steps into one continuous, low intra-method conversion loss process. In the second part, we have established the method of mass spectrometry for allergen quantification of kiwifruits. First, we used the results in the test of Q-ToF to decide the target allergens (Act d 1, Act d 5, and Act d 11) and representative peptides (AD1, AD5, and AD11). Then, we decided the quantitative and qualitative daughter ions of representative peptides on QqQ MS while establishing and optimizing parameters of LC and MS. After validation of our methods, AD1H and AD5H we found to have satisified validation characteristics, including range, accuracy, repeatability, instrumental quantification limit, instrumental detection limit, specificity, and linearity. We have successfully developed and validated mass spectrometry-based allergen quantification method for kiwifruits under the MRM mode. In the third part, we have compared eight common kiwi fruit products to study the influence of different process methods on kiwi allergens. We have determined the allergen contents of Act d 1 and Act d 5 in all samples, which indicated that our method was robust and could be applied to some other common fruit products. We found that allergen contents were significantly reduced under long time heat processing. In conclusion, we have developed two fruit optimized phenol extraction methods, while MPE was combined with rest of the sample preparation methods into one continuous MS sample preparation process. In addition, we have developed a MS-based allergen quantification method for kiwifruits and its derivative products. Our study not only could be an example of analytical method development of food allergen, but also could be applied to improve food safety.