建立液相色谱串联质谱法测定核桃、杏仁、大豆和花生中的蛋白成分,并定量检测核桃露和杏仁露中常见外源成分大豆和花生的含量。样品经蛋白质提取,胰蛋白酶酶解后,采用高效液相色谱-四极杆/静电场轨道阱高分辨质谱 (high performance liquid chromatography coupled with quadrupole-exactive mass spectrometry,HPLC-Q-Exactive-MS)检测,数据经Proteome Discoverer软件与Uniprot蛋白数据库对比分析,筛选出各物种的特征肽段,然后利用高效液相色谱-三重四极杆质谱(high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry,HPLC-QQQ-MS)对核桃、杏仁、大豆和花生的特征多肽进行验证和多反应监测(multiple reaction monitoring, MRM)定量测定。经验证,鉴别方法有良好的专属性和高灵敏度,核桃露和杏仁露中核桃、杏仁、大豆和花生的最低检出限分别为16.3、7.7、1.3和6.8 mg/kg。该方法准确、灵敏,可有效分析植物蛋白饮料中各类蛋白质的来源,对食品植物源性成分的鉴定具有重要意义。
A liquid chromatography-mass spectrometry method was established for the identification of proteins from walnuts, almonds, soybeans and peanuts, and quantitative detection of common exogenous ingredients such as soybean and peanut in walnut drink and almond drink. The samples were determined by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (HPLC-Q-Exactive-MS) after protein extraction and tryptic digestion. The peptide markers of each species were obtained by data analysis with Proteome Discoverer software and Uniprot protein database, and then the verification and multiple reaction monitoring (MRM) of the marker peptides of walnut, almond, soybean and peanut were performed quantitatively using high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QQQ-MS). The results were verified and the identification method had good specificity and high sensitivity. The limit of detection of walnut, almond, soybean and peanut in walnut and almond drink were 16.3 mg/kg, 7.7 mg/kg,1.3 mg/kg and 6.8 mg/kg respectively. This method is accurate and sensitive, and can effectively analyze the origin of proteins in plant protein beverages. And this method is important for identification of plant-derived ingredients in food.
[1] 高艳华,王联结.国内外食品蛋白质发展概况[J].食品研究与开发,2003,24(5):58-60.
[2] 陈贵堂,赵霖.植物蛋白的营养生理功能及开发利用[J].食品工业科技,2004,25(9):137-140.
[3] 丁清龙,曾晓琮,周露,等.广东省植物源性饮料掺假情况摸底调查[J].食品安全质量检测学报,2018,9(12):2 953-2 957.
[4] 水小溪,蔡乐,赵宝华.ELISA技术在食品安全检测中的应用[J].生命科学仪器,2008,6(10):51-53.
[5] 张世伟,赖心田,王石峰,等.基于免疫分析的杏仁蛋白饮料中杏仁蛋白质定量方法研究[J].河南农业科学,2016,45(4):145-149.
[6] GIRISH P S,ANJANEYULU A S R,VISWAS K N,et al.Meat species identification by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)of mitochondrial 12S rRNA gene[J].Meat Science,2005,70(1):107-112.
[7] CALVO J H,OSTA R,ZARAGOZA P.Quantitative PCR detection of pork in raw and heated ground beef and paté[J].Journal of Agricultural and Food Chemistry,2002,50(19):5 265-2 567.
[8] ALI M E,HASHIM U,MUSTAFA S,et al.Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome b gene by TaqMan probe real-time polymerase chain reaction[J].Meat Science,2012,91(4):454-459.
[9] 杨硕,李诗瑶,王鸣秋,等.市售椰子汁(植物蛋白饮料)中椰子、大豆、花生源性成分鉴定的分子生物学方法[J].基因组学与应用生物学,2018,37(10):4 511-4 517.
[10] 郭楠楠,张岩,李永波,等.微滴式数字PCR定量检测杏仁露中杏仁、花生源性成分的分析研究[J].食品科学,2019,40(14):346-351.
[11] 刘津,刘二龙,谢力,等.数字聚合酶链式反应技术在食品安全检测领域的研究应用进展[J].食品科学,2016,37(17):275-280.
[12] HINDSON C M,CHEVILLET J R,BRIGGS H A,et al.Absolute quantification by droplet digital PCR versus analog real-time PCR[J].Nature Methods,2013,10(10):1 003.
[13] 许岩,任皓威,周广运,等.液相色谱质谱联用技术在植物蛋白及多肽研究中的应用[J].食品工业科技,2017,38(17):310-318.
[14] HEICK J,FISCHER M,POPPING B,First screening method for the simultaneous detection of seven allergens by liquid chromatography mass spectrometry[J].Journal of Chromatography A,2011,1218(7):938-943.
[15] RAUNIYAR N.Parallel reaction monitoring:A targeted experiment performed using high resolution and high mass accuracy mass spectrometry[J].Molecular Sciences,2015,16:28 566-28 581.
[16] 古淑青,詹丽娜,赵超敏,等.基于液相色谱-串联质谱法的肉类特征肽段鉴别及掺假测定[J].色谱,2018,36(12):1 269-1 278.
[17] BÖNICK J,HUSCHEK G,RAWEL H M,Determination of wheat,rye and spelt authenticity in bread by targeted peptide biomarkers[J].Journal of Food Composition and Analysis,2017,58(5):82-91.
[18] GU S Q,CHEN N N,ZHOU Y,et al.A rapid solid-phase extraction combined with liquid chromatography-tandem mass spectrometry for simultaneous screening of multiple allergents in chocolates[J].Food Control,2018,84:89-96.
[19] 刘銮雄,李意,阳洪波,等.液相色谱串联质谱法鉴定虾中外源性成分[J].食品工业,2019,40(8):281-284.
[20] 阳洪波,王韦达,李意,等.基于特征肽段的液相色谱-质谱技术鉴定胶原蛋白的物种来源[J].分析测试学报,2018,37(11):1 279-1 286.
