CN116410265B - 一种白唇鹿或白尾鹿鹿源特征多肽及其应用 - Google Patents
一种白唇鹿或白尾鹿鹿源特征多肽及其应用 Download PDFInfo
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Abstract
本发明属于生物检测技术领域,具体涉及一种白唇鹿或白尾鹿鹿源特征多肽及其应用。本发明提供的特征多肽具体为:LLGNVLVVVMAR、FFEHFGDLSSADAVmGNPK。本发明使用高效液相‑三重四级杆质谱联用的方法,能够快速鉴别白尾鹿、白唇鹿的种属来源,能够与马鹿、梅花鹿、驼鹿、驯鹿、黇鹿等区别出来,专属性强,也可应用于其他鹿科动物产品中。本发明通过大量的实验研究,得到了能够进行区别鉴定的特征多肽,专属性强,检测方法简单快捷,提高了白唇鹿、白尾鹿相关产品的质量控制水平,提高了临床用药的安全性和有效性。
Description
技术领域
本发明属于生物检测技术领域,具体涉及一种白唇鹿或白尾鹿鹿源特征多肽及其应用。
背景技术
鹿科动物浑身是宝,鹿茸是重要的中药材,鹿皮为珍贵的装饰品,鹿肉是营养价值丰富的肉类。市场上的鹿科动物制品来源常常无法使用肉眼分辨。
发明内容
针对现有技术中存在的市场空白,本发明提供了一种白唇鹿或白尾鹿鹿源特征多肽。
本发明还提供了一种上述特征多肽在快速鉴别白尾鹿、白唇鹿的种属来源,能够与马鹿、梅花鹿、驼鹿、驯鹿、黇鹿等区别出来,专属性强。
本发明为了实现上述目的所采用的技术方案为:
本发明提供了一种白唇鹿或白尾鹿鹿源特征多肽,所述特征多肽具体为:
肽段1如SEQ ID NO.1所示,具体的序列为:LLGNVLVVVMAR;
肽段2如SEQ ID NO.2所示,具体的序列为:FFEHFGDLSSADAVmGNPK。
进一步的,所述肽段1的检验离子对为:定量离子m/z642.39(z=2)→787.49;定性离子m/z642.39(z=2)→674.40;所述肽段2的检验离子对为:定量离子m/z695.64(z=3)→880.36;定性离子m/z695.64(z=3)→244.17。
本发明还提供了一种白唇鹿或白尾鹿鹿源特征多肽在鹿源种属鉴别中的应用,所述肽段1为白唇鹿专属,当质谱中检出肽段1检验离子对色谱峰,即认为样品为白唇鹿;所述肽段2为白尾鹿专属,当质谱中检出肽段2检验离子对色谱峰,即认为样品为白尾鹿。
进一步的,具体包括以下步骤:
(1)样品粉碎,过筛,称取粉末50mg,加入10mL变性缓冲液,摇匀,置80℃处理过夜,取出放冷至室温,12000r离心10分钟,取500μL样品提取液,使用分子量3kDa的超滤离心管对样品进行脱盐和酶解,即得。
(2)采用高效液相-三重四级杆质谱联用进行鉴定。
本发明所使用的变性缓冲溶液为6M盐酸胍,1M Tris,2.5mM乙二胺四乙酸,加浓盐酸调pH至8.0。
进一步的,步骤(1)中,所述脱盐和酶解的具体步骤为:将样品提取液加入超滤离心管上层,12000r离心10分钟,弃去下层溶液,加入500μL水,12000r离心10分钟,弃去下层溶液,加入500μL水,12000r离心10分钟,弃去下层溶液,加入500μL 1% NH4HCO3溶液和10μL10mg/mL的牛胰蛋白酶溶液,37℃酶解15分钟,取出放冷至室温,离心,取上清液。
本发明鉴定过程中,所述液相条件: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。
所述质谱条件为: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16~20min。
本发明采用特征多肽进行检测时:
判定依据为:
1.肽段1为白唇鹿专属,当质谱中检出肽段1检验离子对色谱峰,即认为样品为白唇鹿。
2.肽段2为白尾鹿专属,当质谱中检出肽段2检验离子对色谱峰,即认为样品为白尾鹿。
判定原则为:
1.样品中检出与白唇鹿对照药材相应的肽段1,则认为样品为白唇鹿来源
2.样品中检出与白尾鹿对照药材相应的肽段2,则认为样品为白尾鹿来源。
本发明的有益效果为:
(1)本发明使用高效液相-三重四级杆质谱联用的方法,能够快速鉴别白尾鹿、白唇鹿-的种属来源,能够与马鹿、梅花鹿、驼鹿、驯鹿、黇鹿等区别出来,专属性强,也可应用于其他鹿科动物产品中。
(2)本发明通过大量的实验研究,得到了能够进行区别鉴定的特征多肽,专属性强,检测方法简单快捷,提高了白唇鹿、白尾鹿相关产品的质量控制水平,提高了临床用药的安全性和有效性。
附图说明
图1 为肽段1 正确序列以及b、y离子归属。
图2 为肽段2 正确序列以及b、y离子归属。
图3为肽段1专属性实验结果。
图4为肽段2专属性实验结果。
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 特征多肽的筛选
样品上清液通过纳升液相色谱-高分辨质谱分析后,将质谱数据导入PEAKS 8.5软件,使用“ Adult-beta globin of deer”蛋白的数据库,进行全部肽段的从头测序及肽段匹配。共从头测序到33185条肽段,数据库匹配,共鉴定到333条肽段。使用高效液相-三重四级杆质谱联用仪验证鉴定到的333条肽段对与马鹿、梅花鹿、驼鹿、驯鹿、白唇鹿、白尾鹿及黇鹿的专属性,考察并选择响应强度最高的母离子与子离子作为定性离子与定量离子。
实施例2
马鹿茸、梅花鹿茸、驼鹿茸、白唇鹿茸、白尾鹿茸、黇鹿茸、驯鹿茸粉碎,过筛,称取粉末50mg,加入10mL变性缓冲液(6M盐酸胍,1M Tris,2.5mM乙二胺四乙酸,加浓盐酸调pH至8.0),摇匀,置80℃处理过夜,取出放冷至室温,12000r离心10分钟,取500μL,使用分子量3kDa的超滤离心管对样品进行脱盐和酶解(12000r离心10分钟,弃去下层溶液,加入500μL水,12000r离心10分钟,弃去下层溶液,加入500μL水,12000r离心10分钟,弃去下层溶液,加入500μL 1% NH4HCO3溶液和10μL牛胰蛋白酶溶液,37℃酶解15分钟,取出放冷至室温,离心,取上清液),即得。
液相条件: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。(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)进样量为5 μL。质谱条件: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16~20min。
图1 为肽段1的对应序列分析图,为肽段1的二级质谱图,其中b离子表示为蓝色线,y离子表示为红色线。图2. 肽段2的对应序列分析图,为肽段2的二级质谱图,其中b离子表示为蓝色线,y离子表示为红色线。图3为肽段1的专属性验证结果,将马鹿、梅花鹿、驼鹿、白唇鹿、黇鹿、驯鹿的鹿茸进行鉴定,发现只有白唇鹿中出现相应色谱峰。图4为肽段2的专属性验证结果,将马鹿、梅花鹿、驼鹿、白唇鹿、黇鹿、驯鹿的鹿茸进行鉴定,发现只有白尾鹿中出相应色谱峰。
对比例1
前期实验中我们发现肽段序列为FFEHFGDLSTPDAVMGNPK的肽段,母离子为708.99,为白唇鹿专属。使用马鹿茸、梅花鹿茸、驼鹿茸、白唇鹿茸、白为鹿茸、黇鹿茸、驯鹿茸对该肽段进行验证后发现,该肽段不仅在白唇鹿茸样品中出峰,在黇鹿茸样品中也出现了响应色谱峰,故认为该肽段不能作为白唇鹿的鉴别肽段。
Claims (8)
1.一种白唇鹿鹿源特征多肽,其特征在于,所述特征多肽具体为:
肽段1的序列为:LLGNVLVVVMAR。
2.一种如权利要求1所述的白唇鹿鹿源特征多肽在鹿源种属鉴别中的应用,其特征在于,所述肽段1为白唇鹿专属,当质谱中检出肽段1检验离子对色谱峰,即认为样品为白唇鹿。
3.根据权利要求2所述的应用,其特征在于,具体包括以下步骤:
(1)样品粉碎,过筛,称取粉末50mg,加入10mL变性缓冲液,摇匀,置80℃处理过夜,取出放冷至室温,12000r离心10分钟,取500μL样品提取液,使用分子量3kDa的超滤离心管对样品进行脱盐和酶解,即得;
(2)采用高效液相-三重四级杆质谱联用进行鉴定。
4.根据权利要求3所述的应用,其特征在于,步骤(1)中,所述变性缓冲溶液为6M盐酸胍,1M Tris,2.5mM乙二胺四乙酸,加浓盐酸调pH至8.0。
5.根据权利要求3或4所述的应用,其特征在于,步骤(1)中,所述脱盐和酶解的具体步骤为:将样品提取液加入超滤离心管上层,12000r离心10分钟,弃去下层溶液,加入500μL水,12000r离心10分钟,弃去下层溶液,加入500μL水,12000r离心10分钟,弃去下层溶液,加入500μL 1% NH4HCO3溶液和10μL 10mg/mL的牛胰蛋白酶溶液,37℃酶解15分钟,取出放冷至室温,离心,取上清液。
6.根据权利要求3所述的应用,其特征在于,所述液相条件:色谱柱为Agilent SB C18,其规格为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。
7.根据权利要求6所述的应用,其特征在于,所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。
8.根据权利要求6或7所述的应用,其特征在于,所述质谱条件为: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16~20min。
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