CN117106031A - 一种驯鹿角、驼鹿角和狍鹿角共有特征肽段及其应用 - Google Patents
一种驯鹿角、驼鹿角和狍鹿角共有特征肽段及其应用 Download PDFInfo
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Abstract
本发明公开了一种驯鹿角、驼鹿角和狍鹿角共有特征肽段及其应用,属于多肽及检测技术领域。所述的驯鹿角、驼鹿角和狍鹿角共有特征肽段氨基酸序列为TGETGASGPP(+15.99)GFAGEK;P(+15.99)为脯氨酸P被氧化修饰为氧化脯氨酸,分子量比脯氨酸增加15.99。应用驯鹿角、驼鹿角和狍鹿角共有特征肽段鉴别鹿角真伪的方法操作简单,灵敏度高,能准确判别鹿角样品是否为驯鹿角、驼鹿角或狍鹿角三种伪品鹿角,检测效率高,为保证鹿角及含鹿角的制剂的质量提供科学方法,对保证鹿角及含有鹿角药品的质量具有重要的意义。
Description
技术领域
本发明属于多肽及检测技术领域,具体涉及一种驯鹿角、驼鹿角和狍鹿角共有特征肽段及其应用。
背景技术
鹿角,为鹿科动物马鹿或梅花鹿已骨化的角或锯茸后翌年春季脱落的角基,具有温肾阳,强筋骨,行血消肿之功效,常用于肾阳不足,阳痿遗精,腰脊冷痛,阴疽疮疡,乳痈初起,瘀血肿痛等病症的治疗。目前市场上正品鹿角不多,伪品非常多,主要是用非正品鹿角如驯鹿角、驼鹿角或狍鹿角冒充使用,该三种鹿角是药材市场上最常见的伪品鹿角,其他伪品鹿角比较少见。
对于养鹿者来说,主要取鹿的鹿茸,因为鹿茸价值远高于鹿角,是主要的经济效益来源,而鹿茸是未骨化的鹿角,所以梅花鹿、马鹿的鹿角未等骨化就被收割,导致正品鹿角产量极低,市场上销售的鹿角及其炮制加工品主要以野外收集自然脱落者或国外进口。进口鹿角以驯鹿角为主,因为雌雄驯鹿均生长鹿角,而其他鹿只有雄鹿才长角,且驯鹿每副鹿角重量较其他鹿角明显偏大,世界范围内野生及饲养的种群也非常大,驯鹿饲养主要用于肉食,也利用其毛皮、鹿乳或用为运输工具,只有部分用于生产驯鹿茸,故驯鹿角产量较高,市场流通量很大。而驼鹿角主要用于加工工艺品,导致产生很多下脚料,其下脚料多冒充正品鹿角使用。狍鹿角以收集自然脱落为主,因狍鹿主要生活在东北密林中,导致收集自然脱落的狍鹿角比较困难,故狍鹿角产量不高,且部分狍鹿角作为工艺品,故药材市场上狍鹿角的流通量不如驯鹿角多。
针对目前市场上驯鹿角、驼鹿角或狍鹿角非正品鹿角冒充正品鹿角使用,但缺乏一种高效鉴别手段,无法对市场伪品鹿角或掺伪鹿角进行快速有效区分,故有效区分伪品鹿角驯鹿角、驼鹿角和狍鹿角就显得至关重要,而通过一种方法将这三种伪品鹿角与正品鹿角同时区别开来就具有重要的意义。
发明内容
针对现有技术中缺乏一次性鉴别鹿角真伪方法的问题,本发明提供了一种驯鹿角、驼鹿角和狍鹿角共有特征肽段及其应用,通过检测驯鹿角、驼鹿角和狍鹿角这三种药材市场上最为常见的伪品鹿角的共有特征肽段,判定鹿角中是否含有驯鹿角、驼鹿角或狍鹿角,为鹿角质量研究提供参考与依据,大大提高鹿角的真伪鉴别效率。
本发明通过以下技术方案实现:
一种驯鹿角、驼鹿角和狍鹿角共有特征肽段,所述的驯鹿角、驼鹿角和狍鹿角共有特征肽段氨基酸序列为TGETGASGPP(+15.99)GFAGEK;所述的P(+15.99)为脯氨酸P被氧化修饰为氧化脯氨酸,结构上增加一个氧原子,分子量比脯氨酸增加15.99。
本发明中,所述的驯鹿角、驼鹿角和狍鹿角共有特征肽段在鉴别鹿角真伪中的应用。
进一步地,鉴别鹿角真伪的方法为,将待检测样品溶液注入液相色谱-高分辨质谱联用仪,获得总离子图,提取待检测样品的一级质谱和二级质谱,以驯鹿角、驼鹿角和狍鹿角共有特征肽段为对照,判断待检测样品溶液中是否含有驯鹿角、驼鹿角或狍鹿角成分。
进一步地,采用电喷雾正离子模式监测,获得350~1550Th范围内的高分辨质谱总离子流图,选择以m/z=739.844±0.005双电荷为检测离子,如果检测出上述离子,且二级质谱中主要碎片离子为721.351±0.005、818.404±0.005、404.214±0.005,则判断待检测鹿角样品中含有驯鹿角、驼鹿角或狍鹿角成分。
进一步地,所述的待检测样品溶液的制备方法为:向20mg待检测样品中加入1mL变性缓冲液,然后加入50μL的 0.5mol/L DTT溶液,置于90℃保温处理4h,取出,放冷至室温,加入120μL的0.55mol/L IAA溶液,摇匀,避光反应60min,离心,取上清液200μL置于10k超滤管中,离心,截留物加200μL的1%碳酸氢铵溶液和5μL胰蛋白酶溶液,涡旋2min,置于37℃恒温培养箱中酶解30min,0.22μm滤膜过滤,得待检测样品溶液。
进一步地,所述的变性缓冲液中包括6mol/L盐酸胍、1.3mol/L Tris和2.4mmol/LEDTA,pH为8.0。
进一步地,液相色谱条件为:Thermo Hypersil GOLD C18色谱柱,100mm×2.1mm,3μm;柱温40℃;流动相A为0.1%甲酸,流动相B为含有0.1%甲酸乙腈,梯度洗脱,进样量为5μL,流速0.3mL/min;
高分辨质谱条件为:Thermo Fusion-Orbitrap高分辨质谱仪,ESI离子源,正离子模式,喷雾电压为2.1kV,离子传输管温度为320℃,S-Lens传输效率设置为60%;一级质谱采用Orbitrap作为质量分析器,采集范围为350~1550Th,二级质谱采用离子阱作为质量分析器,采用HCD碎裂模式,碎裂能量NCE设置为40%;选择m/z=739.844±0.005双电荷作为检测离子。
进一步地,所述梯度洗脱的条件为:0~2 min,5%B;2~75min,5%~45%B;75~80min,45%~100%B;80~85.9min,100%B;85.9~86min,100%~5%B;86~90min,5%B。
本发明取得的有益效果为:
本发明提供了一种驯鹿角、驼鹿角和狍鹿角共有特征肽段,应用于鉴别鹿角真伪,该共有特征肽具有很高的专属性及信号响应;
本发明提供的应用驯鹿角、驼鹿角和狍鹿角共有特征肽段鉴别鹿角真伪的方法操作简单,灵敏度高,能准确判别鹿角样品是否为驯鹿角、驼鹿角或狍鹿角三种伪品鹿角,检测效率高,为保证鹿角及含鹿角的制剂的质量提供科学方法,对保证鹿角及含有鹿角药品的质量具有重要的意义。
附图说明
图1为驯鹿角总正离子流图;
图2为驯鹿角中共有特征肽段的一级质谱图(m/z=739.844±0.005);
图3为驯鹿角中共有特征肽段的二级质谱图;
图4为梅花鹿角总正离子流图;
图5为梅花鹿角中共有特征肽段的一级质谱图(m/z=739.844±0.005);
图6为马鹿角总正离子流图;
图7为马鹿角中共有特征肽段的一级质谱图(m/z=739.844±0.005);
图8为驼鹿角总正离子流图;
图9为驼鹿角中共有特征肽段的一级质谱图(m/z=739.844±0.005);
图10为驼鹿角中共有特征肽段的二级质谱图;
图11为狍鹿角总正离子流图;
图12为狍鹿角中共有特征肽段的一级质谱图(m/z=739.844±0.005);
图13为狍鹿角中共有特征肽段的二级质谱图。
具体实施方式
下面结合具体实施例进一步阐明本发明,应理解这些实施例仅用于说明本发明而不用于限制本发明的范围,在阅读了本发明之后,本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
实施例1
一种驯鹿角、驼鹿角和狍鹿角共有特征肽段,所述的特征肽段序列SEQ ID NO.1为:TGETGASGPP(+15.99)GFAGEK,所述的P(+15.99)为脯氨酸P被氧化修饰为氧化脯氨酸,结构上增加一个氧原子,分子量比脯氨酸增加15.99。
所述的驯鹿角、驼鹿角和狍鹿角共有特征肽段的结构式为:
。
实施例2
(1)取驯鹿角样品粉末20mg,加1mL变性缓冲液(含6mol/L盐酸胍、1.3 mol/L Tris和2.4mmol/L EDTA,pH值为8.0),加50μL的0.5mol/L DTT溶液,置于90℃保温处理4h,取出,放冷至室温,加入120μL的0.55mol/L IAA溶液,摇匀,避光反应60min,离心,取上清液200μL置于10k超滤膜中,离心,截留物加200μL的1%碳酸氢铵溶液和5μL胰蛋白酶溶液(浓度为10mg/mL),涡旋2min,置于37℃恒温培养箱中酶解30min,0.22μm滤膜过滤,即得驯鹿角样品溶液。
(2)将步骤(1)中的驯鹿角样品溶液注入液相色谱=高分辨质谱联用仪,获得驯鹿角样品的一级质谱和二级质谱,以驯鹿角、驼鹿角和狍鹿角共有特征肽段为对照,判断待检测样品溶液中是否含有驯鹿角成分;所述的液相色谱-高分辨质谱联用仪中的液相色谱条件为:Thermo Hypersil GOLD C18色谱柱(100mm×2.1mm,3μm),柱温40℃;流动相A为0.1%甲酸,流动相B为含有0.1%甲酸的乙腈,梯度洗脱:0~2 min,5%B;2~75min,5%B~45%B;75~80min,45%B~100%B;80~85.9min,100%B;85.9~86min,100%B~5%B;86~90min,5%B;进样量为5μL,流速0.3mL/min;
高分辨质谱条件为:Thermo Fusion-Orbitrap高分辨质谱仪,ESI离子源,正离子模式,喷雾电压为2.1 kV,离子传输管温度为320℃,S-Lens传输效率设置为60%。一级质谱采用Orbitrap作为质量分析器,采集范围为350~1550Th;二级质谱采用离子阱作为质量分析器,采用HCD碎裂模式,碎裂能量NCE设置为40%。选择m/z=739.844±0.005(双电荷)作为一级质谱检测离子。
实施例2中驯鹿角总正离子流图如图1所示,驯鹿角中共有特征肽段的一级质谱图(m/z=739.844±0.005)如图2所示,驯鹿角中共有特征肽段的二级质谱图如图3所示,由图可知,在m/z=739.844±0.005(双电荷)范围内可检测出明显色谱峰,且在驯鹿角二级质谱图上述母离子的主要碎片离子为721.353、818.407和404.215,证明该驯鹿角、驼鹿角和狍鹿角共有特征肽段可用来鉴别驯鹿角成分。
实施例3
实施例3中待检测样品为梅花鹿角,检测方法和检测条件与实施例2相同,梅花鹿角总正离子流图如图4所示,梅花鹿角中共有特征肽段的一级质谱图(m/z=739.844±0.005)如图5所示,由图可知,在m/z=739.844±0.005(双电荷)范围内,该样品一级质谱图中均没有检出色谱峰。
实施例4
实施例4中待检测样品为马鹿角,检测方法和检测条件与实施例2相同,马鹿角总正离子流图如图6所示,马鹿角中共有特征肽段的一级质谱图(m/z=739.844±0.005)如图7所示,由图可知,在m/z=739.844±0.005(双电荷)范围内,该样品一级质谱图中均没有检出色谱峰。
实施例5
实施例5中待检测样品为驼鹿角,检测方法和检测条件与实施例2相同,实施例5中驼鹿角总正离子流图如图8所示,驼鹿角中共有特征肽段的一级质谱图(m/z=739.844±0.005)如图9所示,驼鹿角中共有特征肽段的二级质谱图如图10所示,由图可知,在m/z=739.844±0.005(双电荷)范围内可检测出明显色谱峰,且在驼鹿角二级质谱图上述母离子的主要碎片离子为721.352、818.406和404.215,证明该驯鹿角、驼鹿角和狍鹿角共有特征肽段可用来鉴别驼鹿角成分。
实施例6
实施例6中待检测样品为狍鹿角,检测方法与实施例2相同,实施例6中狍鹿角总正离子流图如图11所示,狍鹿角中共有特征肽段的一级质谱图如图12所示,狍鹿角中共有特征肽段的二级质谱图如图13所示,由图可知,在m/z=739.844±0.005(双电荷)范围内可检测出明显色谱峰,且在狍鹿角二级质谱图上述母离子的主要碎片离子为721.354、818.406和404.215,证明该驯鹿角、驼鹿角和狍鹿角共有特征肽段可用来鉴别狍鹿角成分。
综上所述,本发明提供的驯鹿角、驼鹿角和狍鹿角共有特征肽段能够特异性的检测驯鹿角、驼鹿角和狍鹿角,且对梅花鹿角和马路角没有响应,对鹿角的真伪进行鉴别,具有专属性的特点。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.一种驯鹿角、驼鹿角和狍鹿角共有特征肽段,其特征在于,所述的驯鹿角、驼鹿角和狍鹿角共有特征肽段氨基酸序列为TGETGASGPP(+15.99)GFAGEK;
P(+15.99)为脯氨酸P被氧化修饰为氧化脯氨酸,结构上增加一个氧原子,分子量比脯氨酸增加15.99。
2.一种权利要求1所述的驯鹿角、驼鹿角和狍鹿角共有特征肽段在鉴别鹿角真伪中的应用。
3.根据权利要求2所述的驯鹿角、驼鹿角和狍鹿角共有特征肽段在鉴别鹿角真伪中的应用,其特征在于,鉴别鹿角真伪的方法为,将待检测样品溶液注入液相色谱-高分辨质谱联用仪,获得总离子图,提取待检测样品的一级质谱和二级质谱,以驯鹿角、驼鹿角和狍鹿角共有特征肽段为对照,判断待检测样品溶液中是否含有驯鹿角、驼鹿角或狍鹿角成分。
4.根据权利要求3所述的驯鹿角、驼鹿角和狍鹿角共有特征肽段在鉴别鹿角真伪中的应用,其特征在于,采用电喷雾正离子模式监测,获得350~1550Th范围内的高分辨质谱总离子流图,选择以m/z=739.844±0.005双电荷为检测离子,如果检测出上述离子,且二级质谱中主要碎片离子为721.351±0.005、818.404±0.005、404.214±0.005,则判断待检测鹿角样品中含有驯鹿角、驼鹿角或狍鹿角成分。
5.根据权利要求3所述的驯鹿角、驼鹿角和狍鹿角共有特征肽段在鉴别鹿角真伪中的应用,其特征在于,所述的待检测样品溶液的制备方法为:向20mg待检测样品中加入1mL变性缓冲液,然后加入50μL的 0.5mol/L DTT溶液,置于90℃保温处理4h,取出,放冷至室温,加入120μL的0.55mol/L IAA溶液,摇匀,避光反应60min,离心,取上清液200μL置于10k超滤管中,离心,截留物加200μL的1%碳酸氢铵溶液和5μL胰蛋白酶溶液,涡旋2min,置于37℃恒温培养箱中酶解30min,0.22μm滤膜过滤,得待检测样品溶液。
6.根据权利要求3所述的驯鹿角、驼鹿角和狍鹿角共有特征肽段在鉴别鹿角真伪中的应用,其特征在于,所述的变性缓冲液中包括6mol/L盐酸胍、1.3mol/L Tris和2.4mmol/LEDTA,pH为8.0。
7.根据权利要求3所述的驯鹿角、驼鹿角和狍鹿角共有特征肽段在鉴别鹿角真伪中的应用,其特征在于,液相色谱条件为:Thermo Hypersil GOLD C18色谱柱,100mm×2.1mm,3μm;柱温40℃;流动相A为0.1%甲酸,流动相B为含有0.1%甲酸乙腈,梯度洗脱,进样量为5μL,流速0.3mL/min;
高分辨质谱条件为:Thermo Fusion-Orbitrap高分辨质谱仪,ESI离子源,正离子模式,喷雾电压为2.1kV,离子传输管温度为320℃,S-Lens传输效率设置为60%;一级质谱采用Orbitrap作为质量分析器,采集范围为350~1550Th,二级质谱采用离子阱作为质量分析器,采用HCD碎裂模式,碎裂能量NCE设置为40%;选择m/z=739.844±0.005双电荷作为检测离子。
8.根据权利要求7所述的驯鹿角、驼鹿角和狍鹿角共有特征肽段在鉴别鹿角真伪中的应用,其特征在于,所述梯度洗脱的条件为:0~2 min,5%B;2~75min,5%~45%B;75~80min,45%~100%B;80~85.9min,100%B;85.9~86min,100%~5%B;86~90min,5%B。
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