CN107941942B - 一种使用来源于热休克蛋白70的特征多肽鉴别美国肉参的方法 - Google Patents
一种使用来源于热休克蛋白70的特征多肽鉴别美国肉参的方法 Download PDFInfo
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Abstract
本发明涉及一种使用专属性肽段组鉴别美国肉参(Isostichopus badionotus)的方法,以及一组单独或组合使用时,可用来鉴别美国肉参的特征多肽,所述特征多肽为美国肉参蛋白的低同源性多肽片段,所述的低同源性多肽片段指该肽段为美国肉参独有的,且长度在5~30个氨基酸的多肽片段,所述的蛋白虽然发现有部分和植物或细菌等物种和其一致,但对比梅花参、海地瓜、刺参、墨西哥参时,所述特征多肽是美国肉参独有的。
Description
本申请是中国发明申请“CN201610410893.6,一种使用专属性肽段组鉴别美国肉参的方法”的分案申请。
技术领域
本发明属于生物技术领域,具体而言,涉及一种使用专属性肽段组鉴别美国肉参(Isostichopus badionotus)的方法。
背景技术
海参富含酸性黏多糖,对人体有免疫调节剂、抗凝血、控制血糖和降血脂等功效。故此,海参享有“营养宝库”之美誉,现代人把海参誉为“海味八珍之首”。市场上海参种类繁多,不同海参因其营养价值、口感等不同而价格相差巨大。美国肉参是海参的一种,自然生长在冷水海域中,其生长期较长,一般为7-10年,故营养价值极高,除具一般海参拥有的富含蛋白质和多种微量元素的优点外,还具有参体肥壮,发涨率高,参肉厚实,口感爽滑等特点。是烹饪和药用俱佳的极品。
不同品种的海参相比较,有的粘多糖含量高,对心脑血管有好处;有的皂甙含量高,对抗癌有益。海参同人参一样,如果皂甙含量高,则药用价值高,但可能味道不好;而有的海参蛋白质含量高,如茄参和刺参,可以大量用在宾馆和家庭餐桌上,对蛋白质缺乏、营养不良的人群提供身体所需的营养物质。
由于目前缺乏特别有效的识别海参品种的方法,鉴别海参种类的方法还只是靠传统的外观观察,缺乏技术水平的支持,导致不少不良商人以次充好、以假充真欺骗消费者,因此需要一种高效准确的鉴定海参的方法。
发明内容
本发明对美国肉参(Isostichopus badionotus)的肽段进行了研究,建立了从多肽水平对美国肉参进行鉴别的技术,填补了国内外海参鉴别的空白。
本发明首先涉及一组单独或组合使用时,可用来鉴别美国肉参的特征多肽,所述特征多肽为美国肉参蛋白的低同源性多肽片段,所述的低同源性多肽片段指该肽段为美国肉参独有的,且长度在5~30个氨基酸的多肽片段,所述的蛋白为美国肉参中的如下蛋白:
(1)actin isoform 2、histone H2B和glyceraldehyde-3-phosphatedehydrogenase蛋白;
(2)arginine kinase、translationally controlled tumor protein和majoryolk protein 2蛋白;
(3)heat-shock protein 70和heat-shock protein gp96蛋白;
(4)tropomyosin蛋白。
优选的,所述来源于actin isoform 2、histone H2B和glyceraldehyde-3-phosphate dehydrogenase蛋白的特征多肽,其序列为:
SEQ ID NO.1:EIAALAPPTMK;
SEQ ID NO.2:LCYVFLDFEQEMATAASSSSLEK;
SEQ ID NO.3:CDETLFQPSFIGMESAGIHETTYNSIMK;
SEQ ID NO.4:ETYSIYIYK;
SEQ ID NO.5:AGIALSENFVK;
SEQ ID NO.6:VPVPDVSVVDLTLR。
上述肽段1-6的m/z分别为:
571.3;876.1;1069.5;393.9;574.8;754.9。
优选的,所述来源于arginine kinase、translationally controlled tumorprotein和major yolk protein 2蛋白的特征多肽,其序列为:
SEQ ID NO.7:PYDDVLDPAYVISSR;
SEQ ID NO.8:IMDDVLDPAYVISSR;
SEQ ID NO.9:DVITGEELFSDSYK;
SEQ ID NO.10:LVDDLYWR;
SEQ ID NO.11:IGSVFESLNR;
SEQ ID NO.12:PVVFLNPER。
上述肽段7-12的m/z分别为:
855.4;565.3;801.9;540.3;561.3;535.8。
优选的,所述来源于heat-shock protein 70和heat-shock protein gp96蛋白的特征多肽,其序列为:
SEQ ID NO.13:DAGVIAGLQVLR;
SEQ ID NO.14:SEIHELVLVGGSTR;
SEQ ID NO.15:VEIESFFDGEDFSETLTR;
SEQ ID NO.16:DDLVNNLGTIAK;
SEQ ID NO.17:EEAYDYLEPDTIEQLVK;
SEQ ID NO.18:YSQFINFPIYLWGSK。
上述肽段13-18的m/z分别为:
606.4;748.9;1061.0;636.8;1028.0;932.0。
优选的,所述来源于tropomyosin蛋白的特征多肽,其序列为:
SEQ ID NO.19:MSELESQVTSSK;
SEQ ID NO.20:MSELESQVMGSK;
SEQ ID NO.21:QLESELDDTSEK;
SEQ ID NO.22:YDDIEQSSEESER;
SEQ ID NO.23:YNDIEQSSEESER。
上述肽段19-23的m/z分别为:
442.5;442.5;465.2;529.6;529.2。
本发明还涉及一种检测美国肉参的方法,所述方法包括如下步骤:
(1)对待测样本进行质谱前处理,获得待测多肽滤液:
(2)通过质谱法检验待测样本的多肽成分,分析样本中的美国肉参成分。
所述的质谱前处理步骤如下:
(1)称取1g海参样品均质成粉末状态,加入10mL蛋白提取液(8M尿素,50mMNH4HCO3)震荡提取蛋白,高速低温离心(20000r/min),取上清液200ul转移到1ml EP管中;
(2)取2μL1mol/L DTT加入到100μL上述蛋白溶液中37℃反应1小时;
(3)取10μL现配的1mol/L的IAA(配置过程需避光,现用现配)加入到已经冷却至室温的上述反应液中,室温避光反应1小时;
(4)采用10K滤膜20000g超滤30分钟,使用50mmol/L碳酸氢铵溶液反复冲洗滤膜上层,转移至新的EP管中;
(5)再加入碳酸氢铵溶液重复此步骤,合并溶液完成膜下蛋白提取;
(6)取5ul Trypsin酶溶液(1μg/μL的Trypsin酶溶液)加入到上述蛋白溶液中37℃酶解16-18小时;
(7)采用10K滤膜14000g超滤20分钟,收集下层的肽段滤液,待上机检测。
流动相A:0.1%甲酸-乙腈,流动相B:0.1%甲酸-水,
流速:0.25mL/min,
TOF扫描范围:350-1500Da,
正离子反应模式,GS1:35,GS2:45,Curtain Gas:35,ISVF:5500,TEM:500,DP:100,CE:10。
采用AB SCIEX三重四级杆检测,
流动相A:0.1%甲酸-乙腈,流动相B:0.1%甲酸-水,
流速:0.3mL/min,
电喷雾离子源,正离子反应模式,检测方式:MRM,喷雾电压:5500V,离子传输管温度:475℃;鞘气压力:40;辅助气压力:6。
所述的分析样本中的美国肉参成分为,将待测样品的质谱结果对比所述美国肉参蛋白的低同源性多肽片段(特征多肽)的标准质谱谱图,出现所述美国肉参蛋白的低同源性多肽片段的质谱检测谱图时,即可判断该组织样本为美国肉参样本。
优选的,所述的美国肉参蛋白的低同源性多肽片段(特征多肽)为SEQ ID NO.1~23所示的各条特异性多肽。
本发明还涉及使用上述美国肉参蛋白的独有特征多肽鉴别美国肉参的方法以及使用所述特征多肽制备获得的用于鉴别美国肉参的试剂/试剂盒等检测产品。
本发明还涉及所述的特征多肽在制备用于鉴别美国肉参的试剂/试剂盒等检测产品中的应用。
最后,虽然所述的蛋白或特征多肽的部分有植物或细菌等物种和其一致,但仅对比梅花参、海地瓜、刺参、墨西哥参等海参属种时,所述特征多肽是美国肉参独有的。
附图说明
图1,EIAALAPPTMK色谱图
图2,LCYVFLDFEQEMATAASSSSLEK色谱图
图3,CDETLFQPSFIGMESAGIHETTYNSIMK色谱图
图4,ETYSIYIYK色谱图
图5,AGIALSENFVK色谱图
图6,VPVPDVSVVDLTLR色谱图
图7,PYDDVLDPAYVISSR色谱图
图8,IMDDVLDPAYVISSR色谱图
图9,DVITGEELFSDSYK色谱图
图10,LVDDLYWR色谱图
图11,IGSVFESLNR色谱图
图12,PVVFLNPER色谱图
图13,DAGVIAGLQVLR色谱图
图14,SEIHELVLVGGSTR色谱图
图15,VEIESFFDGEDFSETLTR色谱图
图16,DDLVNNLGTIAK色谱图
图17,EEAYDYLEPDTIEQLVK色谱图
图18,YSQFINFPIYLWGSK色谱图
图19,MSELESQVTSSK色谱图
图20,MSELESQVMGSK色谱图
图21,QLESELDDTSEK色谱图
图22,YDDIEQSSEESER色谱图
图23,YNDIEQSSEESER色谱图
具体实施方式
实施例1,特异性多肽的序列来源和比对信息
1.上述多肽SEQ ID NO.1:EIAALAPPTMK;SEQ ID NO.2:LCYVFLDFEQEMATAASSSSLEK;SEQ ID NO.3:CDETLFQPSFIGMESAGIHETTYNSIMK均来自于蛋白actin isoform 2[Holothria glaberrima],其在NCBI上的Accesion number是gi|215882299。经过质谱分析,多肽SEQ ID NO.1:EIAALAPPTMK;SEQ ID NO.2:LCYVFLDFEQEMATAASSSSLEK;SEQ ID NO.3:CDETLFQPSFIGMESAGIHETTYNSIMK在美国肉参中确实存在,且峰形好,强度高。
图1是多肽EIAALAPPTMK在美国肉参中的色谱图,其m/z为571.3。
图2是多肽LCYVFLDFEQEMATAASSSSLEK在美国肉参中的色谱图,其m/z为876.1。
图3是多肽CDETLFQPSFIGMESAGIHETTYNSIMK在美国肉参中的色谱图,其m/z为1069.5。
2.上述多肽SEQ ID NO.4:ETYSIYIYK来自于蛋白histone H2B[Holothuriarubulosa],其在NCBI上的Accesion number是gi|559457。经过质谱分析,多肽SEQ IDNO.4:ETYSIYIYK在美国肉参中确实存在,且峰形好,强度高。
图4是多肽ETYSIYIYK在美国肉参中的色谱图,其m/z为393.9。
3.上述多肽SEQ ID NO.5:AGIALSENFVK;SEQ ID NO.6:VPVPDVSVVDLTLR来自于蛋白glyceraldehyde-3-phosphate dehydrogenase[Apostichopus japonicus],其在NCBI上的Accesion number是gi|334821165。经过质谱分析,多肽SEQ ID NO.5:AGIALSENFVK;SEQID NO.6:VPVPDVSVVDLTLR在美国肉参中确实存在,且峰形好,强度高。
图5是多肽AGIALSENFVK在美国肉参中的色谱图,其m/z为574.8。
图6是多肽VPVPDVSVVDLTLR在美国肉参中的色谱图,其m/z为754.9。
4.经过质谱分析,多肽SEQ ID NO.1-6在梅花参、海地瓜、刺参、墨西哥参中不存在。
实施例2,特异性多肽的序列来源和比对信息
1.上述多肽SEQ ID NO.7:PYDDVLDPAYVISSR;SEQ ID NO.8:IMDDVLDPAYVISSR均来自于蛋白arginine kinase[Apostichopus japonicus],其在NCBI上的Accesion number是gi|4586462。经过质谱分析,多肽SEQ ID NO.7:PYDDVLDPAYVISSR;SEQ ID NO.8:IMDDVLDPAYVISSR在美国肉参中确实存在,且峰形好,强度高。
图7是多肽PYDDVLDPAYVISSR在美国肉参中的色谱图,其m/z为855.4。
图8是多肽IMDDVLDPAYVISSR在美国肉参中的色谱图,其m/z为565.3。
2.上述多肽SEQ ID NO.9:DVITGEELFSDSYK;SEQ ID NO.10:LVDDLYWR来自于蛋白translationally controlled tumor protein[Stichopus monotuberculatus],其在NCBI上的Accesion number是gi|659902918。经过质谱分析,多肽SEQ ID NO.9:DVITGEELFSDSYK;SEQ ID NO.10:LVDDLYWR在美国肉参中确实存在,且峰形好,强度高。
图9是多肽DVITGEELFSDSYK在美国肉参中的色谱图,其m/z为801.9。
图10是多肽LVDDLYWR在美国肉参中的色谱图,其m/z为540.3。
3.上述多肽SEQ ID NO.11:IGSVFESLNR;SEQ ID NO.12:PVVFLNPER来自于蛋白major yolk protein 2[Apostichopus japonicus],其在NCBI上的Accesion number是gi|240846033。经过质谱分析,多肽SEQ ID NO.11:IGSVFESLNR;SEQ ID NO.12:PVVFLNPER在美国肉参中确实存在,且峰形好,强度高。
图11是多肽IGSVFESLNR在美国肉参中的色谱图,其m/z为561.3。
图12是多肽PVVFLNPER在美国肉参中的色谱图,其m/z为535.8。
4.经过质谱分析,多肽SEQ ID NO.7-12在梅花参、海地瓜、刺参、墨西哥参中不存在。
实施例3,特异性多肽的序列来源和比对信息
1.上述多肽SEQ ID NO.13:DAGVIAGLQVLR;SEQ ID NO.14:SEIHELVLVGGSTR;SEQID NO.15:VEIESFFDGEDFSETLTR均来自于蛋白heat shock protein 70[Apostichopusjaponicus],其在NCBI上的Accesion number是gi|194465888。经过质谱分析,多肽SEQ IDNO.113:DAGVIAGLQVLR;SEQ ID NO.14:SEIHELVLVGGSTR;SEQ ID NO.15:VEIESFFDGEDFSETLTR在美国肉参中确实存在,且峰形好,强度高。
图13是多肽DAGVIAGLQVLR在美国肉参中的色谱图,其m/z为606.4。
图14是多肽SEIHELVLVGGSTR在美国肉参中的色谱图,其m/z为748.9。
图15是多肽VEIESFFDGEDFSETLTR在美国肉参中的色谱图,其m/z为1061.0。
2.上述多肽SEQ ID NO.16:DDLVNNLGTIAK;SEQ ID NO.17:EEAYDYLEPDTIEQLVK;SEQ ID NO.18:YSQFINFPIYLWGSK均来自于蛋白heat shock protein gp96[Apostichopusjaponicus],其在NCBI上的Accesion number是gi|355344590。经过质谱分析,多肽SEQ IDNO.16:DDLVNNLGTIAK;SEQ ID NO.17:EEAYDYLEPDTIEQLVK;SEQ ID NO.18:YSQFINFPIYLWGSK在美国肉参中确实存在,且峰形好,强度高。
图16是多肽DDLVNNLGTIAK在美国肉参中的色谱图,其m/z为636.8。
图17是多肽EEAYDYLEPDTIEQLVK在美国肉参中的色谱图,其m/z为1028.0。
图18是多肽YSQFINFPIYLWGSK在美国肉参中的色谱图,其m/z为932.0。
3.经过质谱分析,多肽SEQ ID NO.13-18在梅花参、海地瓜、刺参、墨西哥参中不存在。
实施例4,特异性多肽的序列来源和比对信息
1.上述多肽SEQ ID NO.19:MSELESQVTSSK;SEQ ID NO.20:MSELESQVMGSK;SEQ IDNO.21:QLESELDDTSEK;SEQ ID NO.22:YDDIEQSSEESER;SEQ ID NO.23:YNDIEQSSEESER均来自于蛋白tropomyosin[Apostichopus japonicus],其在NCBI上的Accesion number是gi|302340969。经过质谱分析,多肽SEQ ID NO.19:MSELESQVTSSK;SEQ ID NO.20:MSELESQVMGSK;SEQ ID NO.21:QLESELDDTSEK;SEQ ID NO.22:YDDIEQSSEESER;SEQ IDNO.23:YNDIEQSSEESER在美国肉参中确实存在,且峰形好,强度高。
图19是多肽MSELESQVTSSK在美国肉参中的色谱图,其m/z为442.5。
图20是多肽MSELESQVMGSK在美国肉参中的色谱图,其m/z为442.5。
图21是多肽QLESELDDTSEK在美国肉参中的色谱图,其m/z为465.2。
图22是多肽YDDIEQSSEESER在美国肉参中的色谱图,其m/z为529.6。
图23是多肽YNDIEQSSEESER在美国肉参中的色谱图,其m/z为529.2。
2.经过质谱分析,多肽SEQ ID NO.19-23在梅花参、海地瓜、刺参、墨西哥参中不存在。
实施例5,海参样本处理及检测步骤
对待测海参样本进行分析的步骤包括
(一)样本前处理步骤:
(1)称取1g海参样品均质成粉末状态,加入10mL蛋白提取液(8M尿素,50mMNH4HCO3)震荡提取蛋白,高速低温离心(20000r/min),取上清液200ul转移到1ml EP管中;
(2)取2μL1mol/L DTT加入到100μL上述蛋白溶液中37℃反应1小时;
(3)取10μL现配的1mol/L的IAA(配置过程需避光)加入到已经冷却至室温的上述反应液中,室温避光反应1小时;
(4)采用10K滤膜20000g超滤30分钟,使用200μL 50mmol/L碳酸氢铵溶液反复冲洗滤膜上层,转移至新的EP管中;
(5)再加入200μL碳酸氢铵溶液重复此步骤,合并溶液完成膜下蛋白提取;
(6)取5ul Trypsin酶溶液(1μg/μL的Trypsin酶溶液)加入到上述蛋白溶液中37℃酶解16-18小时;
(7)采用10K滤膜14000g超滤20分钟,收集下层的肽段滤液,待上机检测。
(二)上机检测,
流动相A:0.1%甲酸-乙腈,流动相B:0.1%甲酸-水,
流速:0.25mL/min,
TOF扫描范围:350-1500Da,
正离子反应模式,GS1:35,GS2:45,Curtain Gas:35,ISVF:5500,TEM:500,DP:100,CE:10。
采用AB SCIEX三重四级杆检测,
流动相A:0.1%甲酸-乙腈,流动相B:0.1%甲酸-水,
流速:0.3mL/min,
电喷雾离子源,正离子反应模式,检测方式:MRM,喷雾电压:5500V,离子传输管温度:475℃;鞘气压力:40;辅助气压力:6。
检测结果对比实施例1-4中的各条特异性多肽的质谱谱图,出现实施例1-4所述质谱检测谱图时,即可判断该组织样本为美国肉参样本。
经进一步鉴定,所述肽段为美国肉参专有肽段,在其他海地瓜、墨西哥参、刺参、梅花参等不同海参品种中未见表达。
最后需要说明的是,以上实施例仅用作帮助本领域技术人员理解本发明的实质,并不用作对本发明保护范围的限定。
SEQUENCE LISTING
<110> 山东出入境检验检疫局检验检疫技术中心
<120> 一种使用来源于热休克蛋白70的特征多肽鉴别美国肉参的方法
<160> 23
<170> PatentIn version 3.3
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Claims (3)
1.一种检测美国肉参的方法,所述方法包括如下步骤:
(1)对待测样本进行质谱前处理,获得待测多肽滤液;
(2)通过质谱法检验待测样本的多肽成分,分析待测样本中的美国肉参成分;
所述的分析待测样本中的美国肉参成分为,将待测样品的质谱结果对比序列如SEQ IDNO.13-15所示的各条特异性多肽的质谱谱图,出现与序列如SEQ ID NO.13-15所示的各条特异性多肽的质谱检测谱图相同的谱图时,即可判断该待测样本为美国肉参样本;
所述特征多肽的序列为:
SEQ ID NO.13:DAGVIAGLQVLR;
SEQ ID NO.14:SEIHELVLVGGSTR;
SEQ ID NO.15:VEIESFFDGEDFSETLTR;
所述的质谱前处理步骤如下:
(1)称取海参样品均质成粉末状态,加入蛋白提取液震荡提取蛋白,高速低温离心,取上清液转移到EP管中,所述蛋白提取液为8M尿素、50mM NH4HCO3的溶液;
(2)取DTT加入到上述蛋白溶液中,37℃反应1小时;
(3)取现配的IAA加入到已经冷却至室温的上述反应液中,室温避光反应1小时;
(4)采用10K滤膜超滤30分钟,使用碳酸氢铵溶液反复冲洗滤膜上层,转移至新的EP管中;
(5)再加入碳酸氢铵溶液重复此步骤,合并溶液完成膜下蛋白提取;
(6)取Trypsin酶溶液加入到上述蛋白溶液,37℃酶解16-18小时;
(7)采用10K滤膜超滤20分钟,收集下层的肽段滤液,待上机检测;
所述的质谱法检测为:
采用AB SCIEX5600检测,
流动相A:0.1%甲酸-乙腈,流动相B:0.1%甲酸-水,
流速:0.25mL/min,
TOF扫描范围:350-1500Da,
正离子反应模式,GS1:35,GS2:45,Curtain Gas:35,ISVF:5500,TEM:500,DP:100,CE:10;
或,所述的质谱法检测为:
采用AB SCIEX三重四级杆检测,
流动相A:0.1%甲酸-乙腈,流动相B:0.1%甲酸-水,
流速:0.3mL/min,
电喷雾离子源,正离子反应模式,检测方式:MRM,喷雾电压:5500V,离子传输管温度:475℃;鞘气压力:40;辅助气压力:6。
2.序列如SEQ ID NO.13-15所示的特征多肽在制备用于鉴别美国肉参的试剂或试剂盒中的应用;
所述特征多肽的序列为:
SEQ ID NO.13:DAGVIAGLQVLR;
SEQ ID NO.14:SEIHELVLVGGSTR;
SEQ ID NO.15:VEIESFFDGEDFSETLTR。
3.根据权利要求2所述应用,其特征在于,所述序列如SEQ ID NO.13-15所示的特征多肽与选自以下各组的特征多肽共同应用于制备用于鉴别美国肉参的试剂或试剂盒,
(1)序列如SEQ ID NO.1-3所示的特征多肽:
SEQ ID NO.1:EIAALAPPTMK;
SEQ ID NO.2:LCYVFLDFEQEMATAASSSSLEK;
SEQ ID NO.3:CDETLFQPSFIGMESAGIHETTYNSIMK;
(2)序列如SEQ ID NO.11-12所示的特征多肽:
SEQ ID NO.11:IGSVFESLNR;
SEQ ID NO.12:PVVFLNPER;
(3)序列如SEQ ID NO.19-23所示的特征多肽:
SEQ ID NO.19:MSELESQVTSSK;
SEQ ID NO.20:MSELESQVMGSK;
SEQ ID NO.21:QLESELDDTSEK;
SEQ ID NO.22:YDDIEQSSEESER;
SEQ ID NO.23:YNDIEQSSEESER。
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