超高效液相色谱-串联质谱法同时测定黑巧克力中4种牛奶过敏原蛋白

Determination of four milk allergen proteins in dark chocolates by ultra-performance liquid chromatography-tandem mass spectrometry

目的建立黑巧克力中4种牛奶过敏原蛋白的超高效液相色谱-串联质谱检测方法。方法从Uniprot数据库筛选牛奶主要过敏原蛋白的特异肽段,用Skyline工具模拟胰酶酶切,确定各肽段的多反应监测(multiple reaction monitoring,MRM)方法。50 mmol/L碳酸氢铵(含2 mol/L尿素)提取巧克力中目标蛋白,14000 r/min、4℃离心除脂,胰酶酶解上清液,固相萃取柱净化,Peptide BEH C_(18)(130?,2.1 mm×100 mm,1.7μm)色谱柱分离,以乙腈和水[均含0.1%(V/V)甲酸]为流动相进行梯度分离,标准加入法定量。结果4条特异肽段FFVAPFPEVFGK、NAVPITPTLNR、AVPYPQR和ALPMHIR可分别作为αS1-酪蛋白、αS2-酪蛋白、β-酪蛋白、β-乳球蛋白的定量肽段。其质量浓度与质谱响应峰面积呈现良好线性关系(R^(2)>0.99),方法的检出限和定量限分别为50~100和160~350μg/kg,加标回收率为60.6%~116.7%,相对标准偏差均小于20%(n=6)。结论该方法灵敏、准确、高效,适于检测黑巧克力中4种牛奶过敏原蛋白。

OBJECTIVE To develop a new method for the simultaneous determination of four milk allergen proteins in dark chocolates by ultra-performance liquid chromatography-tandem mass spectrometry.METHODS The specific peptides of main allergen proteins in milk were screened in Uniprot database.Skyline tool was used to simulate the trypsin digestion.Multiple reaction monitoring(MRM)in positive electrospray ionization mode was performed.Milk allergen proteins in chocolates were extracted by 50 mmol/L ammonium bicarbonate(containing 2 mol/L urea)and centrifuged at 14000 r/min with a temperature of 4℃to remove fat.The supernatant was enzymatic hydrolyzed by trypsin for 12 h.The enzymatic hydrolysate was purified by solid-phase extraction.The purified solution was separated on a Peptide BEH C;column(130?,2.1 mm×100 mm,1.7μm)using a gradient mobile phase of acetonitrile and water(both containing 0.1%formic acid(V/V)).Quantitative analysis was made by standard addition method.RESULTS Four specific peptide FFVAPFPEVFGK,NAVPITPTLNR,AVPYPQR and ALPMHIR could be used as quantitative peptides ofαS1-casein,αS2-casein,β-casein andβ-lactoglobulin in chocolates.Good linear relationships between the peak areas of mass spectrometry and the mass concentration could be obtained with correlation coefficients(R;)above 0.99.The limits of detection and quantitation of the above four peptides were in the ranges of 50-100μg/kg and 160-350μg/kg,respectively.The recoveries were ranging from 60.6%to 116.7%with relative standard deviations(n=6)all lower than 20%.CONCLUSION This method is sensitive,accurate and efficient,which is suitable for the analysis of four types of milk allergen proteins in dark chocolate.