CN116284247B - 一种鉴别鹿角胶混淆品的特征多肽及其组合方法、应用 - Google Patents
一种鉴别鹿角胶混淆品的特征多肽及其组合方法、应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及一种鉴别鹿角胶混淆品的特征多肽及其组合方法。本发明提供的9条特征肽段,分别具有不同的专属性,使用特征肽段进行组合,可以鉴别出样品是否是驯鹿角胶、驼鹿角胶、白尾鹿角胶、阿胶、黄明胶、新阿胶、驯鹿皮胶、驼鹿皮胶、白尾鹿皮胶所伪造,该方法专属性高,易于实施,解决的鉴别鹿角胶混淆品来源的难题。
Description
技术领域
本发明属于生物技术领域,具体涉及一种鉴别鹿角胶混淆品的特征多肽及其组合方法。
背景技术
鹿角胶是我国传统名贵中药材,《中国药典》规定其是以马鹿、梅花鹿的鹿角经过反复煎煮熬制获得。由于它的需求量大并且非常名贵,有些不法商家会使用驯鹿角、驼鹿角、白尾鹿角冒充马鹿角或者梅花鹿角作为鹿角胶的原料。另外,由于使用动物的皮类作为原料制胶的得胶率高,还有使用驴皮、牛皮、猪皮、驯鹿皮、驼鹿皮、白尾鹿皮直接熬制,得到的胶用以冒充鹿角胶的现象。传统的薄层色谱、化学鉴别的鉴别方法无法对鹿角胶进行鉴别区分,并且鹿角胶经过反复煎煮熬制,不但失去了原药材的性状特征,其中的遗传物质也被破坏,所以无法使用DNA鉴别的方法鉴别鹿角胶。因此,研发一种能够快速且准确鉴别鹿角胶混淆品的来源的方法成为了亟待解决的问题。
发明内容
针对现有技术中存在的技术空白,本发明提供了一种鉴别鹿角胶混淆品的特征多肽,利用特征多肽作为标志物,其性质稳定易于检测。
本发明还提供了一种上述特征多肽用于鉴别鹿角胶混淆品的组合方法。
本发明为了实现上述目的所采用的技术方案为:
本发明提供了一种鉴别鹿角胶混淆品的特征多肽,所述特征多肽的序列为:
。
本发明利用特征多肽进行鉴别时,判定原则为:
(1)样品中检出pepF,则认为样品为驯鹿角胶;
(2)样品中检出pepG,则认为样品为驼鹿角胶;
(3)样品中检出pepH,则认为样品为白尾鹿角胶;
(4)样品中检出pepA,且未检出pepD,则认为样品为阿胶;
(5)样品中检出pepI,且未检出pepD,则认为样品为黄明胶;
(6)样品中检出pepD,且未检出pepI,则认为样品为新阿胶;
(7)样品中检出pepF,且未检出pepG,则认为样品为驯鹿皮胶;
(8)样品中检出pepE,且未检出pepG,则认为样品为驼鹿皮胶;
(9)样品中检出pepB,且未检出pepC和pepH,则认为样品为白尾鹿皮胶。
本发明还提供了一种上述特征多肽用于鉴别鹿角胶混淆品的组合方法,包括以下步骤:
(1)样品制备:样品粉碎后称取粉末,加入碳酸氢铵溶液摇匀,滤过,取滤液,加入胰蛋白酶溶液,酶解,即得;
(2)采用高效液相-三重四极杆质谱联用进行鉴定。
进一步的,步骤(1)中,所述样品制备的具体过程为:样品粉碎,称取粉末20mg,加入1%(v/v)碳酸氢铵溶液5mL摇匀,滤过,取100μL,置微量进样小瓶中,加入10μL 10mg/ml的胰蛋白酶溶液,37℃酶解3小时,即得。
进一步的,步骤(2)中,所述液相的条件为:色谱柱为Agilent SB C18 (2.1×100mm,1.8μm),柱温43℃,流速0.3mL/min,流动相A为0.1%甲酸溶液,B为0.1%甲酸乙腈溶液,进行梯度洗脱,进样量为5 μL。
优选的,上述梯度洗脱的具体程序为:0~9min,3%B→7.5%B; 9~13min, 7.5%B→25%B; 13~14min,25%B→90%B; 14~17min, 90%B; 17~17.5min, 90%B→97%B, 17.5~21min,97%B。
进一步的,所述质谱的条件为:采用质谱检测器,电喷雾离子化(ESI),正离子模式下,进行多反应监测;鞘气流速46L/hr;辅助气流速850 L/hr;喷雾电压3.5KV;离子源温度150℃;辅助气温度400℃。锥孔电压30V,碰撞电压35V,溶剂延迟(solvent delay)为0~4min和17~21min。
进一步的,所述特征多肽的带电荷数、质核比、以及定性定量离子对具体为:
。
本发明提供的特征肽段是存在于单一物种中作为标志物的肽段,使用特定蛋白酶进行酶解后可以获得。它的性质稳定易于检测,使用质谱采集特征肽段所对应的离子对,如果可以获得响应,则认为样品中含有该物种成分。
本发明提供的9条特征肽段,分别具有不同的专属性,使用特征肽段进行组合,可以鉴别出样品是否是驯鹿角胶、驼鹿角胶、白尾鹿角胶、阿胶、黄明胶、新阿胶、驯鹿皮胶、驼鹿皮胶、白尾鹿皮胶所伪造,该方法专属性高,易于实施,解决的鉴别鹿角胶混淆品来源的难题。
本发明的有益效果为:
(1)本发明提供的特征肽段专属性强,能够有效的解决现有技术中无法鉴别鹿角胶混淆品来源的技术难题。
(2)本发明提供的鉴别方法专属性强,易于实施,样品处理简单。
附图说明
图1为特征多肽的专属性图。
图2为本发明鉴定方法的判定原则图。
图3为特征多肽质谱图。
具体实施方式
下面通过具体的实施例对本发明的技术方案作进一步的解释和说明。
实施例1
选择的特征肽段的序列、带电荷数、质核比、以及定性定量离子对、和肽段来源蛋白如表1所示(其中p为羟脯氨酸)。
表1
具体鉴定过程为:
(1)样品制备方法:样品粉碎,称取粉末20mg,加入1%(v/v)碳酸氢铵溶液5mL摇匀,滤过,取100μL,置微量进样小瓶中,加入10μL 10mg/ml的胰蛋白酶溶液,37℃酶解3小时,即得;
(2)采用高效液相-三重四极杆质谱联用进行鉴定,MRM模式,具体参数为:
液相条件为:色谱柱为Agilent SB C18 (2.1×100mm,1.8μm),柱温43℃,流速0.3mL/min,流动相A为0.1%甲酸溶液,B为0.1%甲酸乙腈溶液,进行梯度洗脱,进样量为5 μL。
上述梯度洗脱的条件为:0~9min,3%B→7.5%B; 9~13min, 7.5%B→25%B; 13~14min,25%B→90%B; 14~17min, 90%B; 17~17.5min, 90%B→97%B, 17.5~21min,97%B。
采用质谱检测器,电喷雾离子化(ESI),正离子模式下,进行多反应监测;鞘气流速46L/hr;辅助气流速850 L/hr;喷雾电压3.5KV;离子源温度150℃;辅助气温度400℃。锥孔电压30V,碰撞电压35V,溶剂延迟(solvent delay)为0~4min和17~21min。
具体检测谱图如图3所示。
特征多肽的专属性图如图1所示;具体判定原则如图2所示,具体为:
(1)样品中检出pepF,则认为样品为驯鹿角胶;
(2)样品中检出pepG,则认为样品为驼鹿角胶;
(3)样品中检出pepH,则认为样品为白尾鹿角胶;
(4)样品中检出pepA,且未检出pepD,则认为样品为阿胶;
(5)样品中检出pepI,且未检出pepD,则认为样品为黄明胶;
(6)样品中检出pepD,且未检出pepI,则认为样品为新阿胶;
(7)样品中检出pepF,且未检出pepG,则认为样品为驯鹿皮胶;
(8)样品中检出pepE,且未检出pepG,则认为样品为驼鹿皮胶;
(9)样品中检出pepB,且未检出pepC和pepH,则认为样品为白尾鹿皮胶。
实施例2
使用发明的方法对市场上20批市售的鹿角胶的进行评价,发现只有8批次鹿角明胶符合规定。检出掺有驯鹿角胶的7批,检出驼鹿角胶的1批,检出白尾鹿角胶的4批,检出新阿胶2批,检出黄明胶1批。具体结果见下表2。
表2
。
Claims (5)
1.一种利用特征多肽鉴别鹿角胶混淆品的组合方法,其特征在于,包括以下步骤:
(1)样品制备:样品粉碎,称取粉末20mg,加入1%(v/v)碳酸氢铵溶液5mL摇匀,滤过,取100μL,置微量进样小瓶中,加入10μL 10mg/ml的胰蛋白酶溶液,37℃酶解3小时,即得;
(2)采用高效液相-三重四极杆质谱联用进行鉴定;
所述特征多肽的序列为:
;
利用特征多肽进行鉴别时,判定原则为:
(1)样品中检出pepF,则认为样品为驯鹿角胶;
(2)样品中检出pepG,则认为样品为驼鹿角胶;
(3)样品中检出pepH,则认为样品为白尾鹿角胶;
(4)样品中检出pepA,且未检出pepD,则认为样品为阿胶或马皮胶;
(5)样品中检出pepI,且未检出pepD,则认为样品为黄明胶;
(6)样品中检出pepD,且未检出pepI,则认为样品为新阿胶;
(7)样品中检出pepF,且未检出pepG,则认为样品为驯鹿皮胶;
(8)样品中检出pepE,且未检出pepG,则认为样品为驼鹿皮胶;
(9)样品中检出pepB,且未检出pepC和pepH,则认为样品为白尾鹿皮胶。
2. 根据权利要求1所述的组合方法,其特征在于,步骤(2)中,所述液相的条件为:色谱柱为Agilent SB C18 :2.1×100mm,1.8μm,柱温43℃,流速0.3mL/min,流动相A为0.1%甲酸溶液,B为0.1%甲酸乙腈溶液,进行梯度洗脱,进样量为5 μL。
3. 根据权利要求2所述的组合方法,其特征在于,所述梯度洗脱的具体程序为:0~9min,3%B→7.5%B;9~13min,7.5%B→25%B;13~14min,25%B→90%B;14~17min,90%B; 17~17.5min, 90%B→97%B;17.5~21min,97%B。
4. 根据权利要求1所述的组合方法,其特征在于,所述质谱的条件为:采用质谱检测器,电喷雾离子化,正离子模式下,进行多反应监测;鞘气流速46L/hr;辅助气流速850 L/hr;喷雾电压3.5KV;离子源温度150℃;辅助气温度400℃;锥孔电压30V,碰撞电压35V,溶剂延迟为0~4min和17~21min。
5.根据权利要求4所述的组合方法,其特征在于,所述特征多肽的带电荷数、质核比、以及定性定量离子对具体为:
。
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