CN117069800B - 一种鉴别东亚钳蝎的特征多肽及其应用 - Google Patents
一种鉴别东亚钳蝎的特征多肽及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及一种鉴别东亚钳蝎的特征多肽及其应用。该特征多肽具体如SEQ ID NO.1‑ SEQ ID NO.4所示;本发明提供的东亚钳蝎特征多肽,具有优异的专属性和稳定性,特异性强,具有良好的应用前景;本发明利用相关特征多肽对相关药品进行鉴别,能够实现对全蝎药材、饮片以及含东亚钳蝎相关药品的质量管控。
Description
技术领域
本发明属于生物技术领域,具体涉及一种鉴别东亚钳蝎的特征多肽及其应用。
背景技术
全蝎为钳蝎科动物东亚钳蝎(Buthus martensii)的干燥体。春末至秋初捕捉,除去泥沙,置沸水或沸盐水中,煮至全身僵硬,捞出,置通风处,阴干。在中医药传统理论中,全蝎被广泛应用于治疗癌症、疮疡肿毒以及中风等疾病,拥有悠久的历史和丰富的临床应用经验。全蝎的主要成分包括多种生物活性物质,如生物碱、多肽、氨基酸、脂肪酸、糖类等。
在现代医学研究中,对全蝎的深入研究与应用也日益受到重视,主要表现在以下几个方面:(1)抗肿瘤研究:全蝎被发现具有显著的抗肿瘤作用,能够抑制癌细胞的生长和增殖,诱导癌细胞凋亡,同时减轻放疗和化疗对正常细胞的损伤。这使得全蝎成为潜在的肿瘤治疗药物;(2)抗炎与免疫调节研究:全蝎具有明显的抗炎作用,能够调节免疫系统的功能,抑制炎症反应,对于自身免疫性疾病等炎症性疾病有一定的治疗潜力;(3)神经保护研究:全蝎中的生物活性成分对神经系统具有保护作用,可减轻脑缺血、脑损伤等神经系统疾病的损伤程度。
因此,全蝎作为传统中药中的重要代表之一,其研究价值日益凸显。对全蝎的深入研究不仅有望为现代医学提供新的治疗思路和方法,而且对于充分挖掘和应用中药资源,推动中医药现代化发展,都具有重要的科学和实践意义。然而,需要进一步加强对全蝎的深入研究,以促进其更广泛地为人类健康服务。
目前,《中国药典》规定全蝎的来源仅为东亚钳蝎,而最近的研究发现,市场上出现了一些其他种类的蝎子如尖刺信使蝎和条斑钳蝎以次充好的现象。这种不合规定的行为不仅损害了消费者的权益,也严重影响了全蝎的质量和疗效。但目前,尚未有可以有效的将东亚钳蝎、尖刺信使蝎和条斑钳蝎进行区分的方法。
发明内容
针对现有技术中存在的问题,本发明提供了一种鉴别东亚钳蝎的特征多肽。
本发明还提供了上述特征多肽在鉴别东亚钳蝎是否为正品中的应用。
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东亚钳蝎的特征多肽,所述特征多肽的序列为:
。
本发明还提供了一种利用上述特征多肽用于鉴别东亚钳蝎是否为正品中的应用,包括以下步骤:
(1)待检测样品预处理:将待检测样品粉碎,称取粉末后加入变性缓冲液和DTT溶液,摇匀,置80℃处理过夜,取出放冷至室温,离心;然后量取上清液加入IAA溶液,避光反应后混匀,离心,然后对样品进行脱盐和酶解,酶解结束后离心,取上清液即得待检测溶液;
(2)使用HPLC-三重四级杆质谱法进行检测,分析对比待检测溶液中的质谱结果,若待检测溶液中在肽段1、肽段2、肽段3、肽段4的出峰处出峰,则认为样品中含有东亚钳蝎。
进一步的,步骤(1)中,待检测样品预处理的具体步骤为:将待检测样品粉碎,称取粉末50mg,加入10mL变性缓冲液和1mL DTT溶液,摇匀,置80℃处理过夜,取出放冷至室温,离心后量取上清液,加入100μL IAA溶液,避光反应30min,混匀,离心,取500μL上清液使用分子量3kDa的超滤离心管对样品进行脱盐和酶解,酶解结束后离心,取上清液即得待检测溶液。
本发明所使用的变性缓冲液为6M盐酸胍,1M Tris,2.5mM乙二胺四乙酸,加浓盐酸调pH至8.0,加水定容;所述DTT溶液的浓度为0.5M;所述IAA溶液的浓度为0.55M。
进一步的,步骤(1)中,所述脱盐和酶解为将样品加入到分子量为3kDa的超滤离心管,12000rpm离心10min,弃去下层溶液,加入500μL水,12000rpm离心10min,弃去下层溶液,加入500μL水,12000rpm离心10min,弃去下层溶液,加入500μL 质量浓度1%的NH4HCO3溶液和10μL 浓度为10mg/mL的牛胰蛋白酶溶液,37℃酶解1小时,取出放冷至室温,于100℃终止酶解反应即可。
进一步的,步骤(2)中,所述HPLC的参数为:色谱柱为 C18:2.1 mm × 100 mm,1.8μm,柱温 43℃,流速0.3mL/min,流动相:A为含0.1%甲酸的水溶液,B为1:1甲醇乙腈溶液,梯度洗脱,进样体积5μL,溶剂延迟为0-4min和26-30min。
进一步的,所述梯度洗脱的程序为:0–12 min,10% B→20% B;12–21 min,20% B→97% B;21–25 min,97% B;25–25.5 min,97% B→10% B;25.5–30 min,10% B。
进一步的,所述质谱的参数为:质谱检测器,电喷雾电离,正离子模式用于多反应监测,鞘气流量46L/h,辅助气体流量850L/h,喷雾电压3.5kV,离子源温度150℃,辅助气体温度400℃,锥孔电压30V,碰撞电压35V。
进一步的,所述肽段1、肽段2、肽段3、肽段4的检测离子对为:
。
本发明的有益效果为:
(1)本发明提供的东亚钳蝎特征多肽,具有优异的专属性和稳定性,特异性强,具有良好的应用前景;
(2)本发明利用相关特征多肽对相关药品进行鉴别,实现对全蝎药材、饮片以及含东亚钳蝎相关药品的质量管控。
附图说明
图1为肽段1的质谱氨基酸序列解析图;
图2为肽段2的质谱氨基酸序列解析图;
图3为肽段3的质谱氨基酸序列解析图;
图4为肽段4的质谱氨基酸序列解析图;
图5为肽段1在样品中的出峰情况图;
图6为肽段2在样品中的出峰情况图;
图7为肽段3在样品中的出峰情况图;
图8为肽段4在样品中的出峰情况图。
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
(1)样品处理方法
分别将东亚钳蝎、尖刺信使蝎和条斑钳蝎样品粉碎,分别称取粉末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),和1mL DTT(0.5M)溶液,摇匀,置80℃处理过夜,取出放冷至室温,离心(12000rpm,10min)。量取上清液500μL,加入100μL IAA(0.55M)溶液,避光反应30min,混匀,离心(12000rpm,10min),取500μL,使用分子量3kDa的超滤离心管对样品进行脱盐和酶解(12000rpm离心10min,弃去下层溶液,加入500μL水,12000rpm离心10min,弃去下层溶液,加入500μL水,12000rpm离心10min,弃去下层溶液,加入500μL 1% NH4HCO3溶液和10μL牛胰蛋白酶溶液(10mg/mL),37℃酶解1小时,取出放冷至室温,于100℃终止酶解反应,离心,取上清液),即得。
(2)纳升液相-高分辨质谱方法
使用纳升液相色谱仪(EASY-nLC 1000,Thermo Scientific,San Jose,CA,USA)结合高分辨率质谱仪(Orbitrap-Fusion,Thermo Scientific,San Jose,CA,USA)对制备的样品进行分析,条件如下:使用 Thermo Acclaim PepMap C18 色谱柱(75 μm × 2 cm、3 μm,Thermo Scientific,San Jose,CA,USA)进行脱盐和富集,使用 Thermo Acclaim PepMapC18 色谱柱(75 μm × 15 cm, 3 μm,Thermo Scientific,San Jose,CA,USA),流速为 300nL/min;流动相A为含2%乙腈的0.1%甲酸水溶液,流动相B为含98%乙腈的0.1%甲酸水溶液,梯度洗脱(0–1 min,1%B→6%B;1– 96min,6%B→22%B;96–113min,22%B→30%B;113–117min,30%B→95%B;117–120min,95%B);进样量为1μL。 高分辨率质谱分析的条件如下。 离子源为Nanospray Flex;分析采用正离子模式,喷雾电压为1,800 V;离子传输毛细管温度为275℃; S-Lens传输效率设置为60%。 初级质谱在分辨率为 60,000、采集范围为 350-1550(m/z) 的 Orbitrap 质量分析仪上进行,二次质谱在采用快速扫描模式进行扫描的Orbitrap 质量分析仪上进行,顶部用于母离子选择的速度数据依赖模式、用于碎片的 HCD模式以及碎片能量 NCE 设置为 40%。
(3)使用PEAKS 8.5处理三种样品质谱数据,进行序列的预测,寻找东亚钳蝎特有、尖刺信使蝎和条斑钳蝎没有的特征多肽作为鉴别方法的特征多肽。东亚钳蝎特有的序列如表1所示。
表1
。
肽段1、肽段2、肽段3和肽段4的质谱氨基酸序列解析如图1-图4所示。
实施例2
使用 HPLC-三重四极杆-质谱法(QTRAP6500 LC/MS,AB SCIEX,Foster City,CA,USA)验证肽生物标志物(肽段1-4)的特异性并建立 MRM 方法。
HPLC 参数设置如下:色谱柱为 C18(2.1 mm × 100 mm,1.8 μm,ZORBAX SBRRHD,Agilent Technologies,Santa Clara,CA,USA),柱温 43℃ ,流速0.3mL/min,流动相A为含0.1%甲酸的水溶液,B为1:1甲醇乙腈溶液,梯度洗脱((0–12 min,10% B→20% B;12–21 min,20% B→97% B;21–25 min,97% B; 25–25.5 min,97% B→10% B;25.5–30 min,10%B) 与进样体积5μL,溶剂延迟为0-4min和26-30min。
质谱参数设置如下:质谱检测器,电喷雾电离(ESI)正离子模式用于多反应监测,鞘气流量46L/h,辅助气体流量850L/h,喷雾电压3.5kV,离子源温度150℃,辅助气体温度400℃,锥孔电压30V,碰撞电压35V。
使用找到的母离子和子离子(如表2)建立方法,鉴别方法:使用东亚钳蝎对照药材和样品,按照实施例1中样品处理方法进行制备,若样品中肽段1-4在对照药材出峰处出峰,则认为样品中含有东亚钳蝎。
表2
。
实施例3 稳定性实验
将制备好的样品在37℃分别放置4h、8h、12h、24h、48h后进HPLC-三重四极杆液相色谱质谱联用仪,使用实施例2所建立的方法结果如下:东亚钳蝎样品中多肽1-4出峰,尖刺信使蝎、条斑钳蝎未出峰。
效果实施例
分别使用东亚钳蝎、尖刺信使蝎、条斑钳蝎样品按照实施例1中样品处理方法处理后进HPLC-三重四极杆质谱联用仪,使用实施例2所建立的方法结果如下:东亚钳蝎样品中多肽1-4出峰,尖刺信使蝎、条斑钳蝎未出峰,具体如图5-图8所示。
Claims (9)
1.一种鉴别东亚钳蝎的特征多肽,其特征在于,所述特征多肽的序列为:
。
2.一种利用如权利要求1所述的特征多肽在用于鉴别东亚钳蝎是否为正品中的应用,其特征在于,包括以下步骤:
(1)待检测样品预处理:将待检测样品粉碎,称取粉末后加入变性缓冲液和DTT溶液,摇匀,置80℃处理过夜,取出放冷至室温,离心;然后量取上清液加入IAA溶液,避光反应后混匀,离心,然后对样品进行脱盐和酶解,酶解结束后离心,取上清液即得待检测溶液;
(2)使用HPLC-三重四级杆质谱法进行检测,分析对比待检测溶液中的质谱结果,若待检测溶液中在肽段1、肽段2、肽段3、肽段4的出峰处出峰,则认为样品中含有东亚钳蝎。
3.根据权利要求2所述的特征多肽在用于鉴别东亚钳蝎是否为正品中的应用,其特征在于,步骤(1)中,待检测样品预处理的具体步骤为:将待检测样品粉碎,称取粉末50mg,加入10mL变性缓冲液和1mL DTT溶液,摇匀,置80℃处理过夜,取出放冷至室温,离心后量取上清液,加入100μL IAA溶液,避光反应30min,混匀,离心,取500μL上清液使用分子量3kDa的超滤离心管对样品进行脱盐和酶解,酶解结束后离心,取上清液即得待检测溶液。
4.根据权利要求3所述的特征多肽在用于鉴别东亚钳蝎是否为正品中的应用,其特征在于,所述变性缓冲液为6M盐酸胍,1M Tris,2.5mM乙二胺四乙酸,加浓盐酸调pH至8.0,加水定容;所述DTT溶液的浓度为0.5M;所述IAA溶液的浓度为0.55M。
5.根据权利要求2或3所述的特征多肽在用于鉴别东亚钳蝎是否为正品中的应用,其特征在于,步骤(1)中,所述脱盐和酶解为将样品加入到分子量为3kDa的超滤离心管,12000rpm离心10min,弃去下层溶液,加入500μL水,12000rpm离心10min,弃去下层溶液,加入500μL水,12000rpm离心10min,弃去下层溶液,加入500μL 质量浓度1%的NH4HCO3溶液和10μL 浓度为10mg/mL的牛胰蛋白酶溶液,37℃酶解1小时,取出放冷至室温,于100℃终止酶解反应即可。
6.根据权利要求2所述的特征多肽在用于鉴别东亚钳蝎是否为正品中的应用,其特征在于,步骤(2)中,所述HPLC的参数为:色谱柱为 C18:2.1 mm × 100 mm,1.8 μm,柱温 43℃,流速0.3mL/min,流动相:A为含0.1%甲酸的水溶液,B为1:1甲醇乙腈溶液,梯度洗脱,进样体积5μL,溶剂延迟为0-4min和26-30min。
7.根据权利要求6所述的特征多肽在用于鉴别东亚钳蝎是否为正品中的应用,其特征在于,所述梯度洗脱的程序为:0–12 min,10% B →20% B;12–21 min,20% B→97% B;21–25min,97% B;25–25.5 min,97% B→10% B; 25.5–30 min,10% B。
8.根据权利要求2所述的特征多肽在用于鉴别东亚钳蝎是否为正品中的应用,其特征在于,所述质谱的参数为:质谱检测器,电喷雾电离,正离子模式用于多反应监测,鞘气流量46L/h,辅助气体流量850L/h,喷雾电压3.5kV,离子源温度150℃,辅助气体温度400℃,锥孔电压30V,碰撞电压35V。
9.根据权利要求1所述的特征多肽在用于鉴别东亚钳蝎是否为正品中的应用,其特征在于,所述肽段1、肽段2、肽段3、肽段4的检测离子对为:
。
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