南极磷虾过敏原的超高效液相色谱-飞行时间质谱方法检测

Determination of Allergens of Antarctic Krill by Ultra-high Performance Liquid Chromatography Coupled with High Resolution Quadrupole Time of Flight Mass Spectrometer

本研究建立了一种基于高分辨质谱的南极磷虾过敏原检测方法:使用Folin-酚法测定蛋白浓度,采用测序级胰蛋白酶对南极磷虾蛋白进行酶解,经超高效液相色谱-飞行时间质谱(UPLC-Q-TOF-MS)对肽段进行分离检测,结合NCBI蛋白数据库和计算机检索对酶解产物进行鉴定,并利用Skyline生物信息学软件进行虚拟酶解,结合三重四极杆质谱对酶解肽段进行验证,鉴定出了南极磷虾中两种主要的过敏原即原肌球蛋白和精氨酸激酶,对应肽段分别为74和44条。该方法灵敏度强,准确度高,操作简便。与传统生物学方法 ELISA和PCR法鉴定过敏原相比大大降低了假阳性结果,直接对过敏原蛋白进行鉴定,提高了检测效率,为食品中过敏原的检测提供了更为有力的手段,也为日后过敏原的消减提供了一定的理论基础。

A method was established in this study to determine allergen of Antarctic krill based on high resolution mass spectrometry. The protein of Euphausia superba is determined by Foline-Phenol and digested by sequence-grade trypsin. The allergens are identified by NCBI protein database and computer retrieval, and validated by using Skyline software combined with triple quadrupole mass spectrometry. Two main kinds of allergens were detected from trpsin digested peptides of Euphausia superba, from which tropomyosin and arginine kinase could be identified. And the corresponding codes of identified peptides of protein allergens were 74 and 44 respectively. As compared with ELISA and PCR, the method is more sensitive, accurate and easy to operate, which may provide a strategy to reduce allergens in foods.