CN116500149A - 一种蚓激酶的超高效液相色谱-串联质谱鉴别方法 - Google Patents
一种蚓激酶的超高效液相色谱-串联质谱鉴别方法 Download PDFInfo
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Abstract
本发明涉及化学分析检测及应用领域,特别涉及一种蚓激酶的超高效液相色谱‑串联质谱鉴别方法。所述方法针对其活性蛋白蚓激酶及纤溶活性蛋白酶,采用超高效液相色谱‑串联质谱方法对两条特征多肽进行分析,特征多肽1氨基酸序列为TSSSNILPDTLQK,特征多肽2氨基酸序列为TGSSNVLPDTLQK。使用上述蚓激酶超高效液相色谱‑串联质谱鉴别方法,采用以下步骤:取待测样品经过胰蛋白酶酶解预处理,取酶解液上清作为供试品溶液;将上述供试品溶液和对照品溶液注入液相色谱‑质谱仪,选择检测离子对,对蚓激酶进行鉴别分析;该方法简便快捷,针对蚓激酶活性蛋白进行检测,提高了蚓激酶鉴别的质量标准及质量控制水平。
Description
技术领域
本发明涉及化学分析检测及应用领域,特别涉及一种蚓激酶的超高效液相色谱-串联质谱鉴别方法。
背景技术
蚓激酶(Lumbrokinase,LK)又称蚯蚓纤溶酶,是从人工养殖的赤子爱胜蚓中提取的一组具有纤溶活性的蛋白水解酶,具有溶解纤维蛋白及血栓的作用。按其组分划分,包括纤维蛋白溶酶、纤维蛋白溶酶原激活物以及类似组织型纤维蛋白溶酶原激活物组分。蚓激酶的适应症为缺血性脑血管病,使过高的纤维蛋白原和血小板凝集率降低,改善症状并防止病情发展。具有溶解纤维蛋白及血栓的作用。蚓激酶虽然有大量相关的研究报道,但其基础研究不够,其物质基础,作用机制和原理,活性位点,蛋白质结构特点,并不清楚。所以蚓激酶在国外还未作为药物上市,仅在日本做为保健品食用。因此蚓激酶的组分和活性研究值得更加深入的研究,同时为提高蚓激酶产品的质量进行控制,能够使其更好地发挥作用。
研究表明蚓激酶成分较为复杂,含有数十种活性相关的蛋白组分,主要包括纤溶酶和蚯蚓激酶,不同企业蚓激酶在种类和数量上略有差异。目前蚓激酶标准中鉴别项目采用紫外光谱和溶血实验法,对最大吸收波长和生物活性进行检测,针对整体蛋白组分。另有报道的纤溶酶谱,针对活性蛋白的数量及相对活力大小进行表征。上述方法均未对具体活性蛋白进行研究,不能对其详细有效组分进行鉴别,开发针对有效蛋白的鉴别方法,对于提高蚓激质量控制水平具有重要意义。
发明内容
为克服现有技术的缺陷,本发明开发了一种蚓激酶的超高效液相色谱-串联质谱鉴别方法。该鉴别方法快速、灵敏,能准确鉴别样品中是否含有蚓激酶活性成分,提高了蚓激酶鉴别的质量标准及质量控制水平。
为实现上述发明目的,本发明采用以下技术方案:
一种蚓激酶的超高效液相色谱-串联质谱鉴别方法,通过高效液相色谱-串联质谱对活性蛋白蚓激酶及纤溶活性蛋白酶的一组特征多肽进行分析,所述特征多肽包括特征多肽1和特征多肽2;其中特征多肽1氨基酸序列为TSSSNILPDTLQK,特征多肽2氨基酸序列为TGSSNVLPDTLQK。
所述的一种蚓激酶的超高效液相色谱-串联质谱鉴别方法,使用上述特征多肽对待测样品中的蚓激酶进行鉴别检测,其特征在于,采用以下步骤:
(1)取待测样品经过酶解预处理,取酶解液上清作为供试品溶液;
(2)取水经过酶解预处理,制备空白溶液;
(3)取步骤(1)及步骤(2)所述的供试品溶液注入液相色谱-质谱仪,采用电喷雾正离子模式,进行多反应监测,以质荷比m/z双电荷702.37→701.38及702.37→814.46作为特征多肽1的检测离子对,以质荷比m/z双电荷680.36→701.38及680.36→814.46作为特征多肽2的检测离子对;
(4)在以步骤(3)所述的检测离子对提取的供试品溶液色谱图中,如能检测到色谱峰且空白溶液出峰处无干扰,则表明待测样品含有氨基酸序列TSSSNILPDTLQK及TGSSNVLPDTLQK,则确定待测样品中含有蚓激酶及纤溶活性蛋白酶。;否则则不含。
进一步地,所述蚓激酶特征多肽为胰蛋白酶酶解预处理。
进一步地,步骤(1)所述经过胰蛋白酶酶解预处理的具体操作为:取待测样品5mg,置10mL量瓶中,用25mmol/L碳酸氢铵溶液溶解并定容;量取200μL,加1mg/mL胰蛋白酶溶液5μL,37℃酶解过夜,90℃灭活10min,放冷至室温,1200rpm离心10min得上清液。
进一步地,步骤(2)所述经过胰蛋白酶酶解预处理的具体操作为:量取200μL25mmol/L碳酸氢铵溶液,加1mg/mL胰蛋白酶溶液5μL,37℃酶解过夜,90℃灭活10min,放冷至室温,1200rpm离心10min得上清液。进一步地,所述液相色谱-质谱仪中液相和质谱检测条件如下:
进一步地,所述液相色谱-质谱仪中液相和质谱检测条件如下:
液相条件:Waters ACQUITY UPLC BEH C18,色谱柱50mm×2.1mm,1.7μm;柱温:40℃;进样量2μL;流速:0.3mL/min;流动相A为0.1%甲酸溶,B为0.1%甲酸乙腈,进行梯度洗脱,洗脱程序:0→2min,流动相A 97%;2→8min,流动相A97%→60%;8.1→10min,流动相A5%→5%;
质谱条件:电喷雾离子源,正离子扫描模式,多反应监测;涡旋离子喷雾温度500℃;离子化电压:5.5kV;碰撞室出口电压10V;入口电压EP 10V;去簇电压DP为80V;特征多肽1碰撞能量CE为35V,特征多肽2碰撞能量CE为45V。
蚓激酶是一种从蚯蚓中提取的复杂酶制剂,含有数十种活性相关的蛋白组分,筛选适宜的的特征肽对相关活性成分进行表征十分重要。本发明筛选的两条特征肽,存在于赤子爱胜蚓中的蚓激酶,蚓激酶-4,蚓激酶-5及纤溶活性蛋白酶-1中,对其检测可同时对几种活性组分蚓激酶及纤溶活性蛋白酶进行检测。
本发明的有益效果:
(1)本发明经过大量实验研究及蛋白质数据库比对,寻找到了蚓激酶制剂中活性成分蚓激酶及纤溶活性蛋白酶,并针对两种活性蛋白中的特征多肽段TSSSNILPDTLQK及TGSSNVLPDTLQK,建立了一种蚓激酶的超高效液相色谱-串联质谱鉴别方法,可用于蚓激酶中活性成分的鉴别检测。
(2)本发明公开的鉴别方法简便快捷,提高了蚓激酶鉴别质量标准,专属性更强,可以大大提高蚓激酶的质量控制水平,确保蚓激酶产品临床用药的有效性和安全性。
附图说明
图1蚓激酶特征多肽1二级质谱图;
图2蚓激酶特征多肽2二级质谱图;
图3专属性考察图谱;
图4 20220101H批次蚓激酶检测图谱;
图5 20220401H批次蚓激酶检测图谱;
图6 20220501H批次蚓激酶检测图谱。
具体实施方式
下面结合具体实施例对本发明进行进一步说明:
下述实施例中相关试剂试药及溶液的制备方法如下:
(1)试剂:胰蛋白酶(Sigma)、蚓激酶(青岛国大药业有限公司)、盐酸胍(VETEC,)、三羟基氨基甲烷(沪市)、二硫苏糖醇(BBI Life Sciences)、碘乙酰胺(Sigma),其它试剂均为分析纯。
(2)25mmol/L碳酸氢铵溶液:称取79.06mg碳酸氢铵,加40mL水溶解。
(3)1mg/mL胰蛋白酶溶液:称取胰蛋白酶10.0mg,加10mL0.1%甲酸水溶解,-20℃保存。
实施例1
蚓激酶活性蛋白及特征多肽的筛选
1仪器设备
Thermo Fusion高分辨质谱仪(Thermo Fisher Scientific,美国),EASY-nLC1000纳升液相色谱仪(Thermo Fisher Scientific,美国),CP225D电子天平(Sartorius,德国),Sigma 3-30K冷冻离心机(Sigma,德国),密理博Milli-QAdvantage A10超纯水仪(Millipore,美国)
2色谱质谱条件
色谱柱为赛默飞Acclaim100 C18纳升色谱柱:75μm×15cm(3μm,/>)和100μm×2cm(5μm,/>)。
流动相A为2%乙腈的0.1%甲酸水溶液;流动相B为98%乙腈的0.1%甲酸水溶液;进样室温度7℃;进样体积2μL。采用EASY-nLC 1000纳升液相系统分离。纳升分离泵流速为300nL/min,梯度洗脱设置见表1。
质谱条件:采用正离子模式进行分析,喷雾电压为2.0kV,离子传输毛细管温度为275℃,S-Lens传输效率设置为60%,采集范围为350-1,500。利用Top speed模式进行母离子选择,采用HCD模式进行碎裂,碎裂能量NCE设置为28%。
表1纳升液相-高分辨质谱梯度洗脱表
3数据采集
取蚓激酶适量配置成1μg/μL;取50μL(1μg/μL)样品溶液,加入75μL变性缓冲液及15μL 1M DTT,60℃反应45min;加入30μL 1M IA,避光反应45min;3k超滤膜脱盐并水洗3次;后加入100μL 25mM NH4HCO3溶解,加1μg胰蛋白酶于37℃过夜酶解,酶解结束后高温灭活并冻干;最后加水500μL复溶并混匀,12000rpm离心10min,取上清于进样瓶中进行液质联用分析。
4.搜库筛选及确定
采用Proteome Discoverer2.5版本对质谱数据进行搜库,条件设置如下:蛋白序列数据库选择uniprot网站(https://www.uniprot.org/)中的赤子爱胜蚓;蛋白酶为胰蛋白酶;最大漏切位点设置为3;肽段长度6-144;肽段母离子质量偏差10ppm,子离子0.02Da;碎片类型为b/y离子;固定化修饰为半胱氨酸甲氧基化(+57.021Da);可变修饰选为甲硫氨酸氧化(+15.995Da);肽段验证是通过设定FDR≤0.01来控制肽段错误率,并选择可信度归属为“高”的肽段。
蚓激酶蛋白组分析结果见表1,主要活性成分为蚓激酶及纤溶蛋白酶,针对两种蛋白,结合实际质谱数据,参照(1)8~25氨基酸、(2)尽量避免易发生人为修饰的肽段、(3)酶切时无漏切位点等原则,结合质谱响应和分离情况,确定“TSSSNILPDTLQK及TGSSNVLPDTLQK”可作为蚓激酶活性成分鉴别的特征多肽。检测结果显示特征多肽的分子量和二级质谱都与理论值相符,见图1和图2。
表2蚓激酶蛋白组分析结果
Accession | Description | MW[kDa] |
A8ILP4 | Lumbrokinase(Fragment) | 24.7 |
Q95V24 | Fibrinolytic enzyme(Fragment) | 19.7 |
Q6T376 | Lumbrokinase-3 | 26.3 |
Q6DKQ2 | Lumbrokinase | 26.3 |
Q8MX72 | Fibrinolytic enzyme component A(Fragment) | 24.8 |
Q308Q8 | Fibrinolytic protease P-III-1(Fragment) | 19.5 |
Q0PGR9 | Fibrinolytic protease(Fragment) | 23 |
A8ILP1 | Lumbrokinase(Fragment) | 24.1 |
Q6T374 | Lumbrokinase-5 | 24.9 |
A0A455RDX9 | Alpha-amylase | 56.7 |
A0A0K2RV70 | 1,3-beta-glucanse | 44.2 |
A0A2Z5VGG0 | Endo-1,4-beta-mannanase | 41.1 |
A0A411K823 | Lysozyme | 23.9 |
D1MJA4 | Superoxide dismutase(Fragment) | 3.6 |
Q8I6N3 | ARSP1 | 25.3 |
A0A1V0JZH1 | Lk | 26.2 |
实施例2
多反应监测(MRM)对蚓激酶活性成分鉴别分析1.仪器设备
SCIEX Triple Quad 6500三重四级杆质谱仪,CP225D电子天平(Sartorius,德国),Sigma 3-30K冷冻离心机(Sigma,德国),密理博Milli-QAdvantage A10超纯水仪(Millipore,美国)。
2.色谱质谱条件
液相条件:Waters ACQUITY UPLC BEH C18,色谱柱50mm×2.1mm,1.7μm;柱温:40℃;进样量2μL;流速:0.3mL/min;流动相A为0.1%甲酸溶,B为0.1%甲酸乙腈,进行梯度洗脱,洗脱程序:0→2min,流动相A 97%;2→8min,流动相A97%→60%;8.1→10min,流动相A5%→5%;
质谱条件:电喷雾离子源,正离子扫描模式,多反应监测;涡旋离子喷雾温度500℃;离子化电压:5.5kV;碰撞室出口电压10V;入口电压EP 10V;去簇电压DP为80V;特征多肽1碰撞能量CE为35V,特征多肽2碰撞能量CE为45V。
3.溶液制备
供试品溶液:取待测样品5mg,置10mL量瓶中,用25mmol/L碳酸氢铵溶液溶解并定容;量取200μL,加1mg/mL胰蛋白酶溶液5μL,37℃酶解过夜,90℃灭活10min,放冷至室温,1200rpm离心10min得上清液。
空白溶液:取200μL 25mmol/L碳酸氢铵溶液,同法操作。
4.测定法
取上述待测溶液各2μL,按照2项下色谱质谱条件检测。
5.专属性考察
取供试品溶液及空白溶液各2μL,进行液质分析,结果空白溶液在出峰位置没有干扰峰出现,供试品溶液特征多肽1出峰时间为5.1min,特征多肽2出峰时间为4.9min,没有其他峰干扰,表明该方法专属性良好,见图3。
6.样品检测
根据上文所述的蚓激酶样品预处理方法以及分析方法,检测结果显示,以质荷比702.37→701.38及702.37→814.46为离子对提取的供试品离子流色谱中,三批蚓激酶样品均能检测到一致的色谱峰(5.1min),均可以检测到特征多肽TSSSNILPDTLQK。以质荷比680.36→701.38及680.36→814.46为离子对提取的供试品离子流色谱中,三批蚓激酶样品均能检测到一致的色谱峰(4.9min)。三批蚓激酶中均可以检测到特征多肽TGSSNVLPDTLQK,上述结果均表明该方法可以对样品中有效成分蚓激酶及纤溶酶进行鉴别。
取10批次含有蚓激酶的样品、10批次同属纤维蛋白溶解药物的尿激酶和10批次空白样品混合放置,随机取样品采用本发明所述方法进行检测,发现使用本发明的处理和分析方法可以百分百检测出含蚓激酶的样品。
以上实施例为本发明较佳的实施方式,单本发明的实施方式并不受实施例的限制,其它任何为背离本发明的精神实质于原理下所做的改变、修饰、组合、替代、简化均应为等效替换方式,都包含在本发明的保护范围之内。
Claims (7)
1.一种蚓激酶的超高效液相色谱-串联质谱鉴别方法,其特征在于,通过高效液相色谱-串联质谱对蚓激酶的一组特征多肽进行分析,所述特征多肽包括特征多肽1和特征多肽2;其中特征多肽1的氨基酸序列为TSSSNILPDTLQK,特征多肽2的氨基酸序列为TGSSNVLPDTLQK。
2.根据权利要求1所述的鉴别方法,其特征在于,所述蚓激酶为活性蛋白蚓激酶或纤溶活性蛋白酶。
3.根据权利要求1或2所述的鉴别方法,使用上述特征多肽对待测样品中的蚓激酶进行鉴别检测,其特征在于,采用以下步骤:
(1)取待测样品经过酶解预处理,取酶解液上清作为供试品溶液;
(2)水经过酶解预处理,制备空白溶液;
(3)取步骤(1)所制备的供试品溶液及步骤(2)所制备的空白溶液注入液相色谱-质谱仪,采用电喷雾正离子模式,进行多反应监测,以质荷比m/z 双电荷702.37→701.38及702.37→814.46作为特征多肽1的检测离子对,以质荷比m/z 双电荷680.36→701.38及680.36→814.46作为特征多肽2的检测离子对,以确定待测样品中是否含有激酶特征多肽中的特征多肽1和特征多肽2;
(4)如果共同检测到特征多肽1和特征多肽2,则证明待测样品中含有蚓激酶;否则则不含有蚓激酶。
4.根据权利要求3所述的鉴别方法,其特征在于,所述酶解预处理为采用胰蛋白酶酶解预处理。
5.根据权利要求3所述的鉴别方法,其特征在于,步骤(1)所述经过酶解预处理的具体操作为:取待测样品5 mg,用25 mmol/L碳酸氢铵溶液溶解并定容至10 mL;量取200 μL,加1mg/mL胰蛋白酶溶液5 μL,37℃酶解过夜,90℃灭活10 min,放冷至室温,1200 rpm离心10min得上清液;
步骤(2)所述水经过酶解预处理的具体操作为:量取200 μL 25 mmol/L碳酸氢铵溶液,加1 mg/mL 胰蛋白酶溶液5 μL,37℃酶解过夜,90℃灭活10 min,放冷至室温,1200 rpm离心10 min得上清液。
6.根据权利要求3所述的鉴别方法,其特征在于,所述液相色谱-质谱仪中液相检测条件如下:Waters ACQUITY UPLC BEH C18,色谱柱 50 mm×2.1 mm,1.7 μm;柱温:40℃;进样量2 μL;流速:0.3 mL/min;流动相A为0.1%甲酸溶,B为0.1%甲酸乙腈,进行梯度洗脱,洗脱程序:0→2 min,流动相A 97%;2→8 min,流动相A97%→60%;8.1→10min,流动相A 5%→5%。
7.根据权利要求3所述的鉴别方法,其特征在于,所述液相色谱-质谱仪中液相和质谱检测条件如下:电喷雾离子源,正离子扫描模式,多反应监测;涡旋离子喷雾温度500℃;离子化电压:5.5 kV;碰撞室出口电压10 V;入口电压EP 10 V;去簇电压DP为80 V;特征多肽1碰撞能量CE为35 V,特征多肽2碰撞能量CE为45V。
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