建立甲殼類過敏原原肌球蛋白之質譜分析方法

海鮮中甲殼類動物是提供人體營養重要來源，然而主要過敏原原肌球蛋白常存在於其加工品與料理中，造成特定族群發生蕁麻疹、氣喘甚至呼吸困難等症狀，為了建立更精確之原肌球蛋白檢測方式，本研究使用具高靈敏性之質譜系統(MS)，藉由同位素稀釋(Isotope dilution)之絕對定量法(Absolute quantification, AQUA)，檢測甲殼類第一過敏原中原肌球蛋白之含量。本實驗以台灣常見蝦種作為研究材料，經胰蛋白酶膠內水解目標蛋白，以電噴灑離子源串聯離子阱質譜儀確認七種蝦子原肌球蛋白之胜肽序列，並透過NCBI、UniProt與Swiss-prot資料庫交叉比對，並選擇峰值強度較強與具甲殼類專一性之片段作為特徵胜肽；在萃取樣品方式，以雙金雞寧酸測定法(Bicinchoninic acid protein assay, BCA)與酵素連結免疫吸附法(Enzyme-linked immunoadsorbent assay, ELISA)法挑選最適化之粗萃取方式；定量方面，用合成特徵胜肽與其氘化同源胜肽作為標準品與內標準品，萃取液經溶液內水解後以電噴灑離子源串聯三段四極柱質譜儀，定量生鮮蝦種與甲殼類加工食品。實驗結果顯示，20mM Tris-HCl+3%NaCl (pH9.0)可萃取最多總蛋白質與原肌球蛋白；使用穩定同位素合成之特徵胜肽，其標準品與氘化內標之母離子質荷比分別為[M+2H]2+= m/z 708.3、[M+2H]2+= m/z 712.3，定量子離子為[M+H]+= m/z 157.0、[M+H]+= m/z 160.0，在不同蝦種中，原肌球蛋白定量結果為555.50-973.93μg/g，確效評估結果顯示，本方法之精密度(CV％)介於2.30-12.80％、回收率介於85.53-101.87％，對雞肉、小卷、鯛魚皆不具交叉反應。本研究建立之質譜分析法除了可應用於不同蝦種與加工品之原肌球蛋白分析，未來甚至可作為甲殼類加工製品是否摻偽之依據。

關鍵字

過敏原；原肌球蛋白；絕對定量法；膠內水解；質譜儀；特徵胜肽；溶液內水解

並列摘要

The crustacean sea foods supply significant nutrition in human body. However, the major allergen “tropomyosin” is often found in processed foods and dishes causing symptoms such as urticaria, asthma, and even dyspnea in specific ethnic groups. In order to establish a more complete method for allergen detection, this study used absolute quantification (AQUA) of isotope dilution to detect first crustacean allergen tropomyosin in highly sensitive mass spectrometry (MS) systems. Seven kinds of Taiwan common shrimps were digested by trypsin using in-gel digestion protocol. The target protein was characterized by electrospray ionization tandem ion trap mass spectrometry. Unique crustacean peptide was regarded as signature peptide matched in different species by NCBI, UniProt and Swiss-prot database. The MS peak of selected signature peptide had strong intensity and separated obviously. In terms of quantification, signature peptide and its deuterated isotopic homologous peptide were as standard and internal standard, respectively. After in-solution digestion, fresh shrimps and crustacean processes foods were analyzed by electrospray ionization tandem triple quadrupole mass spectrometer. The results showed that 20mM Tris-HCl + 3% NaCl (pH 9.0) buffer extracted the most total protein and tropomyosin via BCA and ELISA method, respectively. Using stable-isotope labeled signature peptide fragment which precursor ions mass-to-charge ratio of standard and deuterated internal standard were m/z 708.3 and m/z 712.3 ([M+2H]2+), respectively. The most abundant product ions were m/z 157.0 and m/z 160.0 ([M+H]+) for unlabeled and labeled signature peptides, respectively. The validation of AQUA method was studied using international conference on harmonization protocol. Where 2.30-12.80％for precision(CV％), 85.53-101.87％for recovery rate at three different levels and quality control. The levels of tropomyosin ranged 555.50-973.93μg/g for different shrimp species. This study developed mass spectrometry method that not only applied to analyze for tropomyosin in different shrimp species and processed products, but it can even be used as a basis for whether crustacean processed products are adulterated in the future.