CN112098579A - 一种鹿源特征肽段及其检测方法 - Google Patents
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Abstract
本发明公开了一种鹿源特征肽段及其检测方法,本发明通过大量是实验筛选,研究确定2条鹿源肽段相对含量的比值,并以鹿角胶在混合胶中占比为横坐标,以A肽1/A肽2数值为纵坐标作图,鹿角胶占比与A肽1/A肽2呈线性,建立标准曲线方程为y=4.7903x+0.4106,从而用来区分鹿皮胶与鹿角胶。该方法专属性强、灵敏度高、操作简单,可用于鹿皮胶与鹿角胶的区分及质量控制。从而可以克服现有技术中,鹿角胶与鹿皮胶从外观上难以区分,专属性肽段也难以区分的不足。对保证含有鹿皮胶或鹿角胶的药品、保健食品、健康食品等的质量具有重要意义。
Description
技术领域
本发明涉及一种鹿源特征肽段及其检测方法,具体涉及区分鹿皮胶与鹿角胶,以及鹿角胶中掺有鹿皮胶的样品、掺入鹿皮胶的比例的特征肽段和检测方法。
技术背景
胶类药材包括阿胶、鹿皮胶、鹿角胶、黄明胶等,80%以上的成分为不同类别的胶原蛋白,包括I型胶原α1链(COL1A1)、I型胶原α2链(COL1A2)、II型胶原α1链(COL2A1)、III型胶原α1链(COL3A1)等,其中以COL1A2来源的肽段为主。COL1A2作为高保守蛋白质,广泛存在于不同动物种体内,是构成胶类药材的重要蛋白类成分之一。
鹿角胶与鹿皮胶均来源于梅花鹿或马鹿,两者为名贵胶类中药材,鹿角胶为梅花鹿或马鹿角经水煎煮、浓缩制成的固体胶,鹿皮胶为梅花鹿或马鹿的干燥皮或鲜皮经煎煮、浓缩制成的固体胶。鹿角为马鹿或梅花鹿已骨化的角,价格远高于鹿皮,市场上存在以鹿皮胶掺入鹿角胶以次充好的现象。如何将鹿皮胶与鹿角胶进行区分,着实是胶类药材鉴定研究的难题,给鹿角胶与鹿皮胶鉴别,以及鹿角胶掺入鹿皮胶的伪品鉴别都带来了挑战。
鹿角胶与鹿皮胶从外观上难以区分,且两者均来源于梅花鹿或马鹿,其蛋白质组成相同,因此,通过寻找专属性肽段来区分两者基本不可能。
发明内容:
发明目的:本发明通过大量是实验筛选,研究确定2条鹿源肽段相对含量的比值,从而用来区分鹿皮胶与鹿角胶。该方法专属性强、灵敏度高、操作简单,可用于鹿皮胶与鹿角胶的区分及质量控制。
为实现上述目的,本发明采用如下技术方案:
一种鹿源特征肽段,所述的特征肽为:
肽1:
Gly-Asn-Asp-Gly-Ala-Thr-Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Hyp-Gly-Phe-Hyp-Gly-Ala-Val-Gly-Ala-Lys;
肽2:
Gly-Asn-Asp-Gly-Ala-Thr-Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Hyp-Gly-Phe-Pro-Gly-Ala-Val-Gly-Ala-Lys。
一种鹿源特征肽段的检测方法,包括以下步骤:
(1)将上述的2个鹿源特征肽配成混合对照品溶液;
(2)将待检测鹿皮胶与鹿角胶样品,用胰蛋白酶进行酶切后,将酶解液和步骤(1)鹿源特征肽混合对照品溶液注入液质联用仪,以鹿源特征肽对照,采用ESI正离子模式,多反应监测模式进行检测,选择的离子对包括:肽1:m/z850.4(三电荷)→515.4、肽2:m/z845.0(三电荷)→507.3;以肽1峰面积A肽1与肽2峰面积A肽2的比值确定样品为鹿皮胶或是鹿角胶。
作为优选方案,以上所述的鹿源特征肽段的检测方法,所述的酶切方法包括以下步骤:取待检测胶类药材样品10mg,加入5ml磷酸盐缓冲液,超声使样品完全溶解,12000rpm离心20min,取上清液150μl,置于2ml离心管中,以1ml 50mM PBS稀释,加入适量胰蛋白酶,摇匀,充分酶解,加入10%TFA溶液60μl终止反应,12000rpm离心20min,即得胶类药材酶解液,置于-20℃保存,备用。加入胰蛋白酶的量w/v为0.1%~10%。所述的酶解方式包括:37℃恒温酶解、微波辅助酶解、超声辅助酶解、酶固定化酶解。
作为优选方案,以上所述的鹿源特征肽段的检测方法,液质联用仪检测的液相条件为:色谱柱为1.7μm Waters C18柱,规格为2.1μm×100mm,上样量为2μl,流速0.3ml/min,0~3.5min,10~30%A线性梯度洗脱,3.5~4min,30~10%A线性梯度洗脱,4~6min,10%A洗脱;采用三重四极杆质谱,质谱条件为:m/z 850.4(三电荷)→515.4、m/z 845.0(三电荷)→507.3。
作为优选方案,以上所述的鹿源特征肽段的检测方法,鹿角胶的A肽1/A肽2不得低于5.5,鹿皮胶的A肽1/A肽2不得高于1.2。
本发明提供的鹿皮胶与鹿角胶所含比例的检测方法,其包括以下步骤:
(1)线性关系的建立
将鹿角胶与鹿皮胶按0,10,20,30,40,50,60,70,80,90,100%的比例混合,将每批次混胶样品各取10mg,加入5ml磷酸盐缓冲液,超声使样品完全溶解,12000rpm离心20min,取上清液150μl,置于2ml离心管中,以1ml50mMPBS稀释,加入质量浓度1%胰蛋白酶,摇匀,微波酶解30min,酶解后加入10%TFA溶液60μl终止反应,12000rpm离心20min,即得不同比例混胶样品酶解液,置于-20℃保存,备用;
将不同比例混胶样品酶解液注入液质联用仪,进样量为1μg,液质联用仪检测的液相条件为:色谱柱为1.7μm C18反相色谱柱,规格2.1μm×100mm,流速0.3ml/min,流动相A为乙腈,流动相B为0.1%甲酸,0~3.5min,10~30%A线性梯度洗脱,3.5~4min,30~10%A线性梯度洗脱,4~6min,10%A洗脱;
液质联用仪检测的质谱条件为:电喷雾正离子模式ESI+,质谱参数为:离子源温度500℃;电离电压5500V;脱溶剂温度500℃;离子源气体1,60psi;离子源气体2,60psi;
设置特征肽对应的离子对条件如下:
肽1:m/z 850.4(三电荷)→515.4,DP=162.49,CE=32.87;
肽2:m/z 845.0(三电荷)→507.3,DP=162.52,CE=31.51;
以鹿角胶在混合胶中占比为横坐标,以A肽1/A肽2数值为纵坐标作图,鹿角胶占比与A肽1/A肽2呈线性,建立标准曲线方程为y=4.7903x+0.4106,R2=0.9669;鹿角胶掺入比例与肽1、肽2峰面积呈线性相关,可根据标准曲线方程计算出A肽1与A肽2的比值,确定鹿角胶与鹿皮胶混合比例
有益效果:本发明相比现有技术具有以下优点:
本发明通过大量是实验筛选,研究确定2条鹿源肽段相对含量的比值,并建立的线性方程,从而用来区分鹿皮胶与鹿角胶。该方法专属性强、灵敏度高、操作简单,可用于鹿皮胶与鹿角胶的区分及质量控制。从而可以克服现有技术中,鹿角胶与鹿皮胶从外观上难以区分,专属性肽段也难以区分的不足,取得了非常好的技术进步。
附图说明
图1为鹿角胶在混合胶中占比与A肽1/A肽2数值关系图;
图2为肽1的质谱图;
图3为肽2的质谱图。
具体实施例
下面结合具体实施例对本发明作进一步阐述,但这些实施例不应解释为限制本发明。
实施例1
一种鹿源特征肽,所述的特征肽序列有2个,如序列表1:
肽1:
Gly-Asn-Asp-Gly-Ala-Thr-Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Hyp-Gly-Phe-Hyp-Gly-Ala-Val-Gly-Ala-Lys;
肽2:
Gly-Asn-Asp-Gly-Ala-Thr-Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Hyp-Gly-Phe-Pro-Gly-Ala-Val-Gly-Ala-Lys。
实施例2鹿皮胶肽1与肽2比例关系
取10个批次市售鹿皮胶样品,每批次各取约10mg,加入5ml磷酸盐缓冲液,超声使样品完全溶解,12000rpm离心20min,取上清液150μl,置于2ml离心管中,以1ml 50mM PBS稀释,加入1%胰蛋白酶(w/v),摇匀,37℃恒温酶解12h,酶解后加入10%TFA溶液60μl终止反应,12000rpm离心20min,即得鹿皮胶药材酶解液,置于-20℃保存,备用。
将各批次的鹿皮胶酶解液注入液质联用仪进行检测,进样量为1μg,液质联用仪检测的液相条件为:色谱柱为1.7μm C18反相色谱柱(2.1μm×100mm),流速0.3ml/min,流动相A(乙腈),流动相B(0.1%甲酸),0~3.5min,10~30%A线性梯度洗脱,3.5~4min,30~10%A线性梯度洗脱,4~6min,10%A洗脱。液质联用仪检测的质谱条件为:电喷雾正离子模式(ESI+),质谱参数为:离子源温度500℃;电离电压5500V;脱溶剂温度500℃;离子源气体1,60psi;离子源气体2,60psi。如图2和图3所示质谱图。设置特征肽对应的离子对条件如下:
肽1:m/z 850.4(三电荷)→515.4,DP=162.49,CE=32.87;
肽2:m/z 845.0(三电荷)→507.3,DP=162.52,CE=31.51。
10个批次鹿皮胶A肽1/A肽2数值见表1,A肽1/A肽2平均值为0.612±0.282。
表1鹿皮胶A肽1/A肽2结果
实施例3鹿角胶肽1与肽2比例关系
取10个批次鹿角样品,按照2020版《中国药典》制备鹿角胶方法,获得鹿角胶样品,每批次各取约10mg,加入5ml磷酸盐缓冲液,超声使样品完全溶解,12000rpm离心20min,取上清液150μl,置于2ml离心管中,以1ml 50mM PBS稀释,加入1%胰蛋白酶(w/v),摇匀,超声酶解10min,酶解后加入10%TFA溶液60μl终止反应,12000rpm离心20min,即得鹿角胶酶解液,置于-20℃保存,备用。
将各批次的鹿角胶酶解液注入液质联用仪进行检测,进样量为1μg,液质联用仪检测的液相条件为:色谱柱为1.7μm C18反相色谱柱(2.1μm×100mm),流速0.3ml/min,流动相A(乙腈),流动相B(0.1%甲酸),0~3.5min,10~30%A线性梯度洗脱,3.5~4min,30~10%A线性梯度洗脱,4~6min,10%A洗脱。液质联用仪检测的质谱条件为:电喷雾正离子模式(ESI+),质谱参数为:离子源温度500℃;电离电压5500V;脱溶剂温度500℃;离子源气体1,60psi;离子源气体2,60psi。如图2和图3所示质谱图。设置特征肽对应的离子对条件如下:
肽1:m/z 850.4(三电荷)→515.4,DP=162.49,CE=32.87;
肽2:m/z 845.0(三电荷)→507.3,DP=162.52,CE=31.51。
10个批次鹿角胶A肽1/A肽2数值见表2,A肽1/A肽2平均值为7.428±1.617。
表2鹿角胶A肽1/A肽2结果
实施例4不同比例鹿皮胶与鹿角胶混合样品中肽1与肽2比例关系
将鹿角胶与鹿皮胶按0%,10%,20%,30%,40%,50%,60%,70%,80%,90%,100%的比例混合,每批次混胶样品各取约10mg,分别加入5ml磷酸盐缓冲液,超声使样品完全溶解,12000rpm离心20min,取上清液150μl,置于2ml离心管中,以1ml 50mM PBS稀释,加入1%胰蛋白酶(w/v),摇匀,微波酶解30min,酶解后加入10%TFA溶液60μl终止反应,12000rpm离心20min,即得不同比例混胶样品酶解液,置于-20℃保存,备用。
将不同比例混胶样品酶解液注入液质联用仪,进样量为1μg,液质联用仪检测的液相条件为:色谱柱为1.7μm C18反相色谱柱(2.1μm×100mm),流速0.3ml/min,流动相A(乙腈),流动相B(0.1%甲酸),0~3.5min,10~30%A线性梯度洗脱,3.5~4min,30~10%A线性梯度洗脱,4~6min,10%A洗脱。液质联用仪检测的质谱条件为:电喷雾正离子模式(ESI+),质谱参数为:离子源温度500℃;电离电压5500V;脱溶剂温度500℃;离子源气体1,60psi;离子源气体2,60psi。如图2和图3所示质谱图。设置特征肽对应的离子对条件如下:
肽1:m/z 850.4(三电荷)→515.4,DP=162.49,CE=32.87;
肽2:m/z 845.0(三电荷)→507.3,DP=162.52,CE=31.51。
不同比例混胶样品的A肽1/A肽2数值见表3,鹿角胶在混合胶中占比与A肽1/A肽2数值关系如图1所示。以鹿角胶在混合胶中占比为横坐标,以A肽1/A肽2数值为纵坐标作图,鹿角胶占比与A肽1/A肽2呈线性,y=4.7903x+0.4106,R2=0.9669。表明鹿角胶掺入比例与肽1、肽2峰面积相关,因此可根据A肽1与A肽2的比值确定鹿角胶与鹿皮胶混合比例。
表3鹿角胶/鹿皮胶混合样品掺入比例与峰面积关系
序列表
<110> 南京中医药大学
<120> 一种鹿源特征肽段及其检测方法
<141> 2020-09-01
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Xaa Gly Pro Thr Gly
1 5 10 15
Pro Ala Gly Pro Xaa Gly Phe Xaa Gly Ala Val Gly Ala Lys
20 25 30
<210> 2
<211> 30
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Xaa Gly Pro Thr Gly
1 5 10 15
Pro Ala Gly Pro Xaa Gly Phe Pro Gly Ala Val Gly Ala Lys
20 25 30
Claims (8)
1.一种鹿源特征肽段,其特征在于,所述的特征肽为:
肽1:
Gly-Asn-Asp-Gly-Ala-Thr-Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Hyp-Gly-Phe-Hyp-Gly-Ala-Val-Gly-Ala-Lys;
肽2:
Gly-Asn-Asp-Gly-Ala-Thr-Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Hyp-Gly-Phe-Pro-Gly-Ala-Val-Gly-Ala-Lys。
2.一种鹿源特征肽段的检测方法,其特征在于,包括以下步骤:
(1)将权利要求1所述的2个鹿源特征肽配成混合对照品溶液;
(2)将待检测鹿皮胶与鹿角胶样品,用胰蛋白酶进行酶切后,将酶解液和步骤(1)鹿源特征肽混合对照品溶液注入液质联用仪,以鹿源特征肽对照,采用ESI正离子模式,多反应监测模式进行检测,选择的离子对包括:肽1:m/z 850.4(三电荷)→515.4、肽2:m/z 845.0(三电荷)→507.3;以肽1峰面积A肽1与肽2峰面积A肽2的比值确定样品为鹿皮胶或是鹿角胶。
3.根据权利要求2所述的鹿源特征肽段的检测方法,其特征在于,所述的酶切方法包括以下步骤:取待检测胶类药材样品10 mg,加入5 ml磷酸盐缓冲液,超声使样品完全溶解,12000 rpm离心20 min,取上清液150 μl,置于2 ml离心管中,以1 ml 50 mM PBS稀释,加入适量胰蛋白酶,摇匀,充分酶解,加入10% TFA溶液60 μl终止反应,12000 rpm离心20 min,即得胶类药材酶解液,置于-20℃保存,备用。
4.根据权利要求3所述的鹿源特征肽段的检测方法,其特征在于,加入胰蛋白酶的量w/v为0.1%~10%。
5.根据权利要求3所述的鹿源特征肽段的检测方法,其特征在于,酶解方式包括:37℃恒温酶解、微波辅助酶解、超声辅助酶解、酶固定化酶解。
6.根据权利要求2所述的鹿源特征肽段的检测方法,其特征在于,液质联用仪检测的液相条件为:色谱柱为1.7μm Waters C18柱,规格为2.1μm × 100 mm,上样量为2μl,流速0.3ml/min,0~3.5 min,10~30%A线性梯度洗脱,3.5~4 min,30~10%A线性梯度洗脱,4~6min,10%A洗脱;采用三重四极杆质谱,质谱条件为:m/z 850.4(三电荷)→515.4、m/z 845.0(三电荷)→507.3。
7.根据权利要求2所述的鹿源特征肽段的检测方法,其特征在于,鹿角胶的 A肽1/A肽2不得低于5.5,鹿皮胶的 A肽1/A肽2不得高于1.2。
8.鹿皮胶与鹿角胶所含比例的检测方法,其特征在于,包括以下步骤:
(1)线性关系的建立
将鹿角胶与鹿皮胶按0,10,20,30,40,50,60,70,80,90,100%的比例混合,将每批次混胶样品各取10 mg,加入5 ml磷酸盐缓冲液,超声使样品完全溶解,12000 rpm离心20 min,取上清液150 μl,置于2 ml离心管中,以1 ml 50 mM PBS稀释,加入质量浓度1%胰蛋白酶,摇匀,微波酶解30 min,酶解后加入10% TFA溶液60 μl终止反应,12000 rpm离心20 min,即得不同比例混胶样品酶解液,置于-20℃保存,备用;
将不同比例混胶样品酶解液注入液质联用仪,进样量为1 μg,液质联用仪检测的液相条件为:色谱柱为1.7μm C18反相色谱柱,规格2.1 μm × 100 mm,流速0.3ml/min,流动相A为乙腈,流动相B为0.1%甲酸,0~3.5 min,10~30%A线性梯度洗脱,3.5~4 min,30~10%A线性梯度洗脱,4~6 min,10%A洗脱;
液质联用仪检测的质谱条件为:电喷雾正离子模式ESI+,质谱参数为:离子源温度500℃;电离电压5500V;脱溶剂温度500℃;离子源气体1,60psi;离子源气体2,60psi;
设置特征肽对应的离子对条件如下:
肽1:m/z 850.4(三电荷)→515.4,DP=162.49, CE=32.87;
肽2:m/z 845.0(三电荷)→507.3,DP=162.52, CE=31.51;
以鹿角胶在混合胶中占比为横坐标,以A肽1/A肽2数值为纵坐标作图,鹿角胶占比与A肽1/A肽2呈线性,建立标准曲线方程为y = 4.7903x + 0.4106,R² = 0.9669;鹿角胶掺入比例与肽1、肽2峰面积呈线性相关,可根据标准曲线方程计算出A肽1与A肽2的比值,确定鹿角胶与鹿皮胶混合比例。
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