CN114539377A - 一种使用液相色谱-质谱检测十足目水产过敏原的方法 - Google Patents
一种使用液相色谱-质谱检测十足目水产过敏原的方法 Download PDFInfo
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Abstract
本发明涉及一种使用液相色谱‑质谱检测十足目水产过敏原的方法,所述方法包括,(1)对待测样本进行蛋白组测定前处理,获得待测多肽滤液;(2)检测十足目水产物种中的共有特征多肽组,通过液相色谱‑质谱法测定样品中的多肽组成分;(3)如样品中含有SEQ ID NO.1~6所述的多肽组,则样品中含有十足目水产主要过敏原。
Description
技术领域
本发明属于检测技术领域,具体的,涉及一种使用液相色谱-质谱检测十足目水产过敏原的方法。
背景技术
世界上的甲壳动物的种类很多,大约2.6万种之多。其中甲壳亚门软甲纲十足目分类下的虾、蟹等水产营养丰富,味道鲜美,具有很高的经济价值。但是甲壳亚门十足目水产也属于国际认定的八大食物过敏原之一,随着海产品受到越来越多人的青睐,此类食物过敏也受到重视。研究表明,水产品中的原肌球蛋白(TM)被鉴定为十足目水产的主要过敏原。其相对分子质量介于34kDa~39kDa之间,由两条多肽链构成,是虾、蟹等水产品的重要抗原,具有高度保守的氨基酸序列,在不同品种之间有着较高的同源性。
近年来,食物过敏发病率在全球呈现逐年升高趋势,现已成为全球公共卫生问题;世界卫生组织指出,全球有22%-25%的人患有过敏性疾病,且人数逐年增加,其中食物过敏占很大部分。世界变态反应组织报告显示,全世界30%~40%的人被过敏困扰,过敏已成为全球第六大疾病。曾经有研究者进行了严重过敏反应的研究,发现:食物诱因占过敏性休克患者的77%,药物占7%,昆虫占0.6%,其余15%不明原因。可见食物过敏问题不容忽视,且针对食物过敏,目前国内外尚无有效的根治措施,可通过避免摄入或接触相关食物过敏原来预防食物过敏的发生。但是由于现代饮食中食物准备方法的复杂性及加运输过程中外来过敏原的污染,许多患者会意外接触到已知的过敏原从而危害身体健康。
针对于十足目水产过敏原的检测,目前主要的检测方法有ELISA、免疫层析和PCR检测技术,其中PCR检测方法具有较高的灵敏度,但该方法通过检测DNA来达到检测过敏原的目的属于间接检测,且易受脂类、表面活性剂或乳化剂等基质干扰,从而导致假阴结果,ELISA和免疫层析检测方法的特异性取决于抗原、抗体的制备,由于存在交叉反应会导致假阴假阳结果的出现,两种检测方法目前均存在检测试剂盒,其中免疫层析技术无法定量,不同厂家的ELISA试剂盒差异较大,定量结果之间往往缺乏较好的可比性,这限制了其应用范围。
针对上述问题,液相色谱-质谱法结合蛋白质组学对食物过敏原进行检测鉴定,在未来过敏原检测领域具有广阔的应用前景。相比于上述检测技术,质谱检测技术更稳定、更准确、灵敏度更高,而且质谱法弥补了PCR不能直接检测过敏原蛋白的短板以及免疫学方法存在的通量低和交叉干扰的弊端,能够对蛋白消化后的多肽进行明确鉴定和定量,并且可以同时检测多种过敏原蛋白,实现过敏原的高通量检测,提高检测效率。目标过敏原的定量检测主要采用多反应监测(MRM)方式,其主要优势是靶向分析中的高灵敏度、高选择性和高重现性,能够在复杂的样品中对微量靶蛋白质进行精确测定。
由于目前现有过敏原检测技术均存在着一定的短板,因此需要一种高效准确的十足目水产过敏原检测方法。
发明内容
基于此,提出本发明。
本发明首先涉及一组多肽组在鉴定十足目水产主要过敏原中的应用,所述的多肽组的氨基酸序列及核质比(m/z)为:
SEQ ID NO.1:SLEVSEEK,460.73;
SEQ ID NO.2:LEDELVNEK,544.78;
SEQ ID NO.3:LAEASQAADESER,688.82;
SEQ ID NO.4:LAMVEADLER,573.79;
SEQ ID NO.5:IVELEEELR,565.31;
SEQ ID NO.6:LNTATTK,374.71。
本发明还涉及一种检测样品中是否含有十足目水产主要过敏原原肌球蛋白的方法,所述方法包括如下步骤:
(1)对待测样本进行蛋白组测定前处理,获得待测多肽滤液;
(2)检测十足目水产物种中的共有特征多肽组,通过液相色谱-质谱法测定样本中的多肽组成分;
(3)如样品中含有SEQ ID NO.1~6所述的多肽组,则样品中含有十足目水产主要过敏原。
其中,
步骤(1)所述的对待测样本进行蛋白组测定前处理,获得待测多肽滤液的步骤如下:
1)样品均质成肉糜,加入丙酮萃取,待上层丙酮呈淡黄色或无色时,过滤洗涤,将沉淀风干后,粉碎成丙酮粉;
2)称取述丙酮粉,加入蛋白提取液(0.05M Tris-HCL pH8.2,0.2M KCL,50mM DTT)震荡提取蛋白15分钟,高速低温离心(4℃,10000r/min,5min),取上清液过0.22μL无菌滤膜,得蛋白提取液;优选的,丙酮粉和蛋白提取液的比例为1:20(w/v);
3)取上述蛋白提取液转移到10kDa的超滤管中,将1M二硫苏糖醇(DTT)溶液加入到上述蛋白提取液中还原二硫键;优选的,DTT和蛋白提取液的比例为1:50(v/v);
4)将1M碘代乙酰胺(IAA)溶液加入到上述步骤3)的反应液中,反应1小时;优选的,IAA和反应液的比例为5:52(v/v);
5)将上述反应后液体超滤20分钟,使用50mM碳酸氢铵溶液反复冲洗滤膜上层,转移至新的EP管中;
6)再加入200μL 50mM碳酸氢铵溶液复溶膜上蛋白,得蛋白组溶液;
7)按照蛋白质质量:酶质量=50:1的比例,将胰蛋白酶溶液加入到上述蛋白组溶液中37℃酶解16-18小时;
8)采用10kDa滤膜12000r/min超滤20分钟,收集下层的肽段滤液。
步骤(2)所述的检测十足目水产物种中的共有特征多肽组,通过液相色谱-质谱法测定样本中的多肽组成分的方法为:
1)采用液相色谱-四级杆/飞行时间质谱检测,
液相色谱条件:
流动相A:0.1%甲酸-水,流动相B:0.1%甲酸-乙腈,梯度为,
流速:0.25mL/min,
质谱条件:
TOF扫描范围:350-1500Da,
正离子反应模式,GS1:60,GS2:50,Curtain Gas:40,ISVF:5500,TEM:525,DP:100,CE:10;
或2)采用液相色谱-三重四级杆检测,
流动相A:0.1%甲酸-水,流动相B:0.1%甲酸-乙腈,梯度为,
流速:0.3mL/min,
进样体积:5μL,
电喷雾离子源,正离子反应模式,检测方式:MRM,喷雾电压:5000V,离子传输管温度:475℃;气帘气压力:45;碰撞气压力:8;喷雾气压力:40;辅助加热气压力:55。
本发明的有益效果是:
1、本发明以十足目水产主要过敏原蛋白原肌球蛋白为检测基础,基于高分辨质谱鉴定分析了的酶切肽段,依据十足目水产物种中原肌球蛋白序列的高度相似性及保守性,挖掘到6条肽段作为十足目水产物种的一组特异性肽段,其响应度高、稳定性高、特异性高。
2、根据筛选到的特征肽段的离子对,建立十足目水产物种特征肽段LC-MS/MS-MRM方法定性检测样品中的十足目水产过敏原蛋白成分。
3、实际应用中,将待测样品按同样方法进行前处理和液相色谱-质谱分析分析,能够在肉制品、水产品、调味料、酱料中有良好的应用效果。
附图说明
图1,天然原肌球蛋白DDA分析总离子流色谱图;
图2,天然原肌球蛋白DIA分析总离子流色谱图;
图3,天然原肌球蛋白特征肽段MRM总离子流图;
图4,合成肽段MRM总离子流图;
图5,各特征肽段提取离子流图;
图6,特异性验证结果1;
图7,特异性验证结果2;
图8,特异性验证结果3。
图6-8中,1:牛肉 2:猪肉 3:鸡肉 4:羊肉 5:大黄花鱼 6:鳕鱼 7:大菱鲆 8:蛤蜊9:海螺 10:美人贝 11:缢蛏 12:牡蛎 13:小鱿鱼 14:杂色鲍 15:扇贝 16:淡水小龙虾17:口虾蛄 18:波士顿龙虾 19:中华绒螯蟹 20:三疣梭子蟹 21:中国对虾 22:南美白对虾,图6-8中涉及的样品均购买于山东省青岛市城阳区上马利客来购物超市。
上图中,各个肽段的三字母简称对应表3所列十足目水产主要过敏原原肌球蛋白特征肽段;
图1-3所用天然原肌球蛋白样品来自中国海洋大学食品安全实验室;
图4-5所述合成肽段由中国合肥国肽生物科技有限公司合成,纯度均>95%;
图4-5中的不同名称的肽段对应的氨基酸序列及核质比(m/z)分别为:
SLE:SEQ ID NO.1:SLEVSEEK,460.73;
LED:SEQ ID NO.2:LEDELVNEK,544.78;
LAE:SEQ ID NO.3:LAEASQAADESER,688.82;
LAM:SEQ ID NO.4:LAMVEADLER,573.79;
IVE:SEQ ID NO.5:IVELEEELR,565.31;
LNT:SEQ ID NO.6:LNTATTK,374.71。
具体实施方式
实施例1、对待测样本进行蛋白组测定前处理
对待测样本进行蛋白组测定前处理,具体步骤如下:
(1)称取6g十足目水产物种和其他软体动物、脊椎动物、双壳贝类等物种的肌肉组织,样品均质成肉糜,加入30mL预冷(-20℃)丙酮,除去脂肪和色素,带上层丙酮呈淡黄色或无色时,过滤洗涤,将沉淀于通风橱风干后,使用粉碎机将其制备成丙酮粉。
(2)称取0.15g上述丙酮粉,加入3mL蛋白提取液(0.05M Tris-HCL pH8.2,0.2MKCL,50mM DTT)震荡提取蛋白15分钟,高速低温离心(4℃,10000r/min,5min),取上清液过0.22μL无菌滤膜,得蛋白提取液。
(3)取上述蛋白提取液400μL转移到500μL10kDa的超滤管(外管为2mL EP管)中,取8μL 1M二硫苏糖醇(DTT)溶液加入到400μL上述蛋白提取液中,37℃反应1小时还原二硫键;
(4)取40μL 1M碘代乙酰胺(IAA)溶液,配置过程需避光,现用现配,加入到已冷却至室温的上述反应液中,室温避光反应1小时;
(5)将上述反应后液体,进行离心,12000r/min超滤20分钟,使用50mM碳酸氢铵溶液反复冲洗滤膜上层,转移至新的EP管中;
(6)再加入200μL 50mM碳酸氢铵溶液复溶膜上蛋白,得蛋白组溶液;
(7)按照蛋白质质量:酶质量=50:1的比例,将胰蛋白酶溶液加入到上述蛋白组溶液中37℃酶解16-18小时;
(8)采用10kDa滤膜12000r/min超滤20分钟,收集下层的肽段滤液,待上机检测。
实施例2、通过液相色谱-质谱法联用测定蛋白组成分,
液相色谱-质谱法联用的方法为,
(1)采用液相色谱-四级杆/飞行时间质谱检测,
液相色谱条件:
流动相A:0.1%甲酸-水,流动相B:0.1%甲酸-乙腈,梯度见表1,
表1、UHPLC梯度洗脱程序
流速:0.25mL/min,
质谱条件:
TOF扫描范围:350-1500Da,
正离子反应模式,GS1:60,GS2:50,Curtain Gas:40,ISVF:5500,TEM:525,DP:100,CE:10;
(2)采用液相色谱-三重四级杆检测,
流动相A:0.1%甲酸-水,流动相B:0.1%甲酸-乙腈,梯度见表2,
表2液相梯度洗脱程序
流速:0.3mL/min,
进样体积:5μL,
电喷雾离子源,正离子反应模式,检测方式:MRM,喷雾电压:5000V,离子传输管温度:475℃;气帘气压力:45;碰撞气压力:8;喷雾气压力:40;辅助加热气压力:55。
(3)搜库鉴定高分辨质谱获得的wiff数据导入Proteinpilot软件,并以NCBI中的十足目水产物种原肌球蛋白为蛋白数据库搜索鉴定酶切肽段序列。参数设置:样品类型:identification;半胱氨酸(Cys);烷基化试剂:Iodoacetic acid;消化方式:Trypsin;允许生物学修饰和氨基酸替代;搜索事项:Thorough ID;可信阈值(Unused Protscore(conf)):>1.3(10.0%)。结果选取Unused>1.5、响应高、氨基酸个数7~20、Conf>99%、无漏切无修饰的肽段作为预选特征肽段。同时在NCBI数据库BLAST比对验证肽段的生物学特异性,筛选肽段如表3所示。
表3、十足目水产主要过敏原原肌球蛋白特征肽段组
(4)构建十足目水产过敏原TM定性检测液相色谱-三重四级杆-MRM方法,色谱条件:流动相A:0.1%甲酸-水,流动相B:0.1%甲酸-乙腈,梯度见表3,流速:0.3mL/min,进样体积:5μL,质谱条件:电喷雾离子源,正离子反应模式,检测方式:MRM,喷雾电压:5000V,离子传输管温度:475℃;气帘气压力:45;碰撞气压力:8;喷雾气压力:40;辅助加热气压力:55。
(5)离子对参数设置根据Skyline软件来预测,根据Proteinpilot软件的鉴定肽段结果,利用Skyline软件构建肽段,MRM方法的母子离子对及碰撞能量等。每个肽段筛选3个特征离子对,结果如表4所示。
表4十足目水产过敏原原肌球蛋白各特征肽段MRM参数及保留时间列表
(6)特异性验证选用牛肉、猪肉、鸡肉、羊肉、大黄花鱼、鳕鱼、大菱鲆、蛤蜊、海螺、美人贝、缢蛏、牡蛎、小鱿鱼、杂色鲍、扇贝、淡水小龙虾、口虾蛄、波士顿龙虾、中华绒螯蟹、三疣梭子蟹、中国对虾、南美白对虾经相同前处理后,进行实际样品的特异性验证。结果见图6-8;可见,选定的6条特征多肽,能够很好的区分十足目水产和其他样品。
最后需要说明的是,以上实施例仅用于帮助本领域技术人员理解本发明的实质,不用于限定本发明的保护范围。
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Claims (5)
1.一组特征多肽组在鉴定十足目水产主要过敏原中的应用,所述的多肽组的氨基酸序列及核质比(m/z)分别为:
SEQ ID NO.1:SLEVSEEK,460.73;
SEQ ID NO.2:LEDELVNEK,544.78;
SEQ ID NO.3:LAEASQAADESER,688.82;
SEQ ID NO.4:LAMVEADLER,573.79;
SEQ ID NO.5:IVELEEELR,565.31;
SEQ ID NO.6:LNTATTK,374.71。
2.一种检测样品中是否含有十足目水产主要过敏原TM的方法,所述方法包括如下步骤:
(1)对待测样本进行蛋白组测定前处理,获得待测多肽滤液;
(2)检测十足目水产物种中的共有特征多肽,通过液相色谱-质谱法测定样本中的多肽成分;
(3)如样品中含有权利要求1所述的特征多肽组,则样品中含有十足目水产主要过敏原。
3.根据权利要求2所述的方法,其特征在于,步骤(1)所述的对待测样本进行蛋白组测定前处理,获得待测多肽滤液的步骤如下:
1)样品均质成肉糜,加入丙酮萃取,待上层丙酮呈淡黄色或无色时,过滤洗涤,将沉淀风干后,粉碎成丙酮粉;
2)称取述丙酮粉,加入蛋白提取液(0.05M Tris-HCL pH8.2,0.2M KCL,50mM DTT)震荡提取蛋白15分钟,高速低温离心(4℃,10000r/min,5min),取上清液过0.22μL无菌滤膜,得蛋白提取液;优选的,丙酮粉和蛋白提取液的比例为1:20(w/v);
3)取上述蛋白提取液转移到10kDa的超滤管中,将1M二硫苏糖醇(DTT)溶液加入到上述蛋白提取液中还原二硫键;优选的,DTT和蛋白提取液的比例为1:50(v/v);
4)将1M碘代乙酰胺(IAA)溶液加入到上述步骤3)的反应液中,反应1小时;优选的,IAA和反应液的比例为5:52(v/v);
5)将上述反应后液体超滤20分钟,使用50mM碳酸氢铵溶液反复冲洗滤膜上层,转移至新的EP管中;
6)再加入200μL 50mM碳酸氢铵溶液复溶膜上蛋白,得蛋白组溶液;
7)按照蛋白质质量:酶质量=50:1的比例,将胰蛋白酶溶液加入到上述蛋白组溶液中37℃酶解16-18小时;
8)采用10kDa滤膜12000r/min超滤20分钟,收集下层的肽段滤液。
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115963273A (zh) * | 2022-10-09 | 2023-04-14 | 中国海洋大学 | 一种三疣梭子蟹nSCP或rSCP的应用与试剂盒 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109557193A (zh) * | 2018-07-09 | 2019-04-02 | 中国检验检疫科学研究院 | 一种芝麻主要过敏原的质谱定性检测方法 |
US20190302081A1 (en) * | 2016-07-21 | 2019-10-03 | Nisshin Seifun Group Inc. | Allergen detection method |
CN110531019A (zh) * | 2019-09-25 | 2019-12-03 | 南京农业大学 | 一种基于不同动物源性肉类特征多肽的肉样掺假定量检测方法 |
-
2022
- 2022-02-25 CN CN202210181213.3A patent/CN114539377A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190302081A1 (en) * | 2016-07-21 | 2019-10-03 | Nisshin Seifun Group Inc. | Allergen detection method |
CN109557193A (zh) * | 2018-07-09 | 2019-04-02 | 中国检验检疫科学研究院 | 一种芝麻主要过敏原的质谱定性检测方法 |
CN110531019A (zh) * | 2019-09-25 | 2019-12-03 | 南京农业大学 | 一种基于不同动物源性肉类特征多肽的肉样掺假定量检测方法 |
Non-Patent Citations (2)
Title |
---|
JIANHUA WANG ET AL: "Quantification of crustacean tropomyosin in foods using high-performance liquid chromatography–tandem mass spectrometry method", J SCI FOOD AGRIC, vol. 101, pages 1 - 2 * |
PANYARAT LAURCHAN ET AL: "Molecular Characterization and Cross-Allergenicity of Tropomyosin from Freshwater Crustaceans", J. AGRIC. FOOD CHEM., vol. 69, pages 3 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115963273A (zh) * | 2022-10-09 | 2023-04-14 | 中国海洋大学 | 一种三疣梭子蟹nSCP或rSCP的应用与试剂盒 |
CN115963273B (zh) * | 2022-10-09 | 2024-04-12 | 中国海洋大学 | 一种三疣梭子蟹nSCP或rSCP的应用与试剂盒 |
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