反相液相色谱-串联质谱法筛选牙鲆鱼蛋白质中潜在的过敏原

Screening potential allergens in Paralichthys olivaceus by reverse phase liquid chromatography-tandem mass spectrometry

中文关键词:  牙鲆鱼  过敏原  液相色谱-串联质谱法  序列同源性分析

英文关键词:Paralichthys olivaceus  allergen  reverse phase liquid chromatography-tandem mass spectrometry  sequence homology analysis

中文摘要:

      目的 建立反相液相色谱-串联质谱法(reversed phase liquid chromatography coupled with tandem mass spectrometry, RPLC-MS/MS)筛选牙鲆鱼(Paralichthys olivaceus)中潜在过敏原。方法 采用RPLC-MS/MS技术对牙鲆鱼的蛋白质样品进行蛋白质组学分析。再将牙鲆鱼蛋白质样品在体外模拟胃液中消化, 通过十二烷基硫酸钠-聚丙烯酰胺凝胶(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)电泳观察不同酶解时间下鱼蛋白的酶解情况, 将耐酶解的蛋白通过胶内酶解处理后采用RPLC-MS/MS法进行分析。最后将鉴定到的蛋白质与已知过敏原进行序列同源性分析。结果 在SDS-PAGE中酶解时间为3 h时位于40 kDa处的条带仍清晰可见, 此处的蛋白为耐酶解的蛋白质,经胶内酶解后共鉴定到5种蛋白质。与已知过敏原蛋白进行蛋白序列同源性分析, 牙鲆鱼的原肌球蛋白alpha-1与已知过敏原Ore m 4的序列相似度较高。2种蛋白质的一致性和相似性分别为96.5%和97.9%。甘油醛-3-磷酸脱氢酶与已知过敏原Tri a 34序列对比一致性和相似性分别为72%和83%。肌酸激酶 1与已知过敏原Pen m 2序列对比的一致性和相似性分别为42%和63%, 2组序列相似度大于35%, 说明也具有相似性。结论 耐胃蛋白酶酶解的大部分蛋白质是潜在的过敏原, 其序列与其他物种已知的蛋白过敏原高度相似。该方法可作为筛选食物中潜在过敏原的一种新方法。

英文摘要:

      Objective To establish a method for screening potential allergens in Paralichthys olivaceus by reverse phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS). Methods Proteomic analysis of proteins from the Paralichthys olivaceus was performed using RPLC-MS/MS. The Paralichthys olivaceus protein sample was digested in simulated gastric juice in vitro, and the fish protein was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the case of enzymatic hydrolysis, the enzymatically digested protein was subjected to enzymatic hydrolysis and analyzed by RPLC-MS/MS. Then, sequence homology analysis between identified proteins and known allergens was performed. Results The band at 40 kDa was still clearly visible in SDS-PAGE at 3 h, and the protein here was an enzymatically resistant protein. Five proteins were identified after in-gel digestion. Homology analysis of the protein sequence with known allergen proteins showed that the sequence similarity of tropomyosin alpha-1 in flounder was higher than that of known allergen Ore m 4. The consistency and similarity of the two proteins were 96.5% and 97.9%, respectively. The comparison consistency and similarity of the sequences of glyceraldehyde 3-phosphate dehydrogenase and the known allergen Tri a 34 were 72% and 83%, respectively. The consistency and similarity of Pen m 2 sequence between creatine kinase 1 and known allergen were 42% and 63%, respectively. The similarity of 2 groups of sequences was greater than 35%, indicating that they were also similar. Conclusion Most proteins resistant to pepsin enzymatic hydrolysis are potential allergens whose sequences are highly similar to those of known proteins in other species. This method can be used as a new method for screening potential allergens in food.