CN116814588B - 圆斑蝰蛇磷脂酶a2特征多肽及其应用 - Google Patents
圆斑蝰蛇磷脂酶a2特征多肽及其应用 Download PDFInfo
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Abstract
本发明涉及化学分析定性检测技术领域，尤其涉及圆斑蝰蛇磷脂酶A2特征多肽及其应用。所述特征多肽由特征多肽氨基酸序列为LIDAKQEAFSFFK。该特征多肽可用于检测待测样品中圆斑蝰蛇磷脂酶A2特征多肽种属来源，检测准确度高，可以显著提高蛇毒血凝酶注射液的质量控制水平，确保产品临床用药的有效性和安全性。另一方面也为圆斑蝰蛇磷脂酶A2更多药理学活性和临床应用的开发提供了保障。
Description
技术领域
本发明涉及化学分析定性检测技术领域，尤其涉及圆斑蝰蛇磷脂酶A2特征多肽及其应用。
背景技术
磷脂酶A2(phospholipase A2，PLA2)几乎存在于所有蛇毒液中，是生物体膜上一种重要的含钙金属酶,能特异性地水解sn-3-磷酸甘油酯2位上酯酰键生成游离的脂肪酸和溶血磷脂，广泛参与多种生物进程如磷脂代谢、免疫防御和信号传导，因而成为研究脂肪酸代谢、磷脂膜结构、磷脂与膜蛋白相互作用以及膜蛋白结构与功能的重要工具酶。圆斑蝰(Daboia russelii siamensis)，是蛇亚目蝰科蝰亚科蝰属下的一种有毒蝰蛇。研究表明圆斑蝰脂酶A2具有神经毒性、间接溶血，强肌坏死活性和细胞毒性。不同种属来源磷脂酶A2氨基酸序列存在差异，导致作用位点不同，表现出不一样的药理活性。目前市场中蛇毒血凝酶注射液的有效成分是从蝰蛇科蛇毒中提取的蛇毒血凝酶，可用于各种出血疾病。磷脂酶A2作为潜在的杂质，对制剂中磷脂酶控制对于提高药品质量，保证临床用药安全十分重要。目前磷脂酶A2检测多采用卵磷脂降解法检测磷脂酶活性，该方法操作复杂，灵敏度较低。因此开发一种简便快捷、具有种属鉴别的作用的方法对圆斑蝰蛇磷脂酶A2研究及蛇毒血凝酶注射液的质量控制十分重要。
液质联用技术拥有高灵敏度、高分辨率、高稳定性、高特异性和高效分离等特点，广泛应用于样品的定性定量分析之中。对于蛋白类样品的种属鉴别，重要是特征肽的筛选、前处理及质谱条件的优化，特征肽需要同时具有种属特异性、稳定性、质谱响应高等，后续针对该特征肽进行相应的前处理及液质条件优化。
发明内容
为解决上述技术问题，本发明的发明目的是提供圆斑蝰磷脂酶A2特征多肽，所述特征多肽能够在表征样品中磷脂酶A2的种属来源中发挥重要作用，填补了圆斑蝰磷脂酶A2种属鉴别的空白。
为实现上述发明目的，本发明采用以下技术方案：
圆斑蝰蛇磷脂酶A2特征多肽，所述特征多肽氨基酸序列为LIDAKQEAFSFFK。
一种上述圆斑蝰蛇磷脂酶A2特征多肽的应用，所述特征多肽可用于鉴别磷脂酶A2的种属来源。
进一步地，上述鉴别方法采用以下步骤：
(1)取待测样品溶解，进行酶解灭活后作为供试品溶液；
(2)水经过酶解处理，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 772.41→294.18及772.41→342.20作为特征多肽的检测离子对，以确定待测样品中是否含有圆斑蝰蛇磷脂酶A2特征多肽中的特征多肽；
(4)如果检测到上述特征多肽,则证明待测样品中含有磷脂酶A2且来源于圆斑蝰蛇；反之若则证明待测样品中不含有来源于圆斑蝰蛇的磷脂酶A2。
进一步地，步骤(3)所述液相色谱-质谱仪中液相和质谱检测条件中，液相条件为：Waters ACQUITY UPLC BEH C18色谱柱(50mm*2.1mm，1.7μm)；流动相A：0.1％甲酸溶液流动相，B：0.1％甲酸乙腈；柱温：40℃；进样量：5μL；流速：0.3ml/min；按下表进行梯度洗脱：
进一步地，步骤(1)的具体操作为：
①待测样品溶解：待测样品用1％碳酸氢铵配制为浓度1μg/μL；
②酶解：取步骤①溶解后样品100μL，加2μg蛋白酶于37℃过夜酶解，酶解结束后高温灭活；
③离心：最后取步骤②酶解灭活的样品，12000rpm离心10min，取上清液即得供试品溶液。
优选地，所述的蛋白酶为胰蛋白酶。
与现有技术相比，本发明具有以下有益效果：由于已知白眉蝮蛇、矛头蝮蛇、尖吻蝮蛇等蛇毒的磷脂酶A2氨基酸序列中不含有本发明所述的特征多肽，因此，该特征多肽可用于检测待测样品中磷脂酶A2特征多肽的种属来源，检测准灵敏度高，一方面填补了蛇毒血凝酶注射液质量标准的空白，可以显著提高其质量控制水平，确保产品临床用药的有效性和安全性；另一方面也为圆斑蝰蛇磷脂酶A2更多药理学活性和临床应用的开发提供了保障。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。以下，结合附图来详细说明本发明的实施方案，其中：
图1为实施例1中圆斑蝰蛇磷脂酶A2特征多肽WDIYPYSWK Blast结果；
图2为实施例1中圆斑蝰蛇磷脂酶A2特征多肽WDIYPYSWK二级质谱图；
图3为实施例2中矛头蝮蛇蛇毒特征多肽提取离子色谱图；
图4为实施例2中圆斑蝰蛇蛇毒特征多肽提取离子色谱图。
具体实施方式
在接下来的描述中进一步阐述了本发明的具体细节用于充分理解本发明。本发明中的说明书所使用的术语只是为了用于说明本发明的优点和特点，不是旨在于限制本发明。
除非另行定义，本发明中所使用的所有专业与科学术语属于本发明的技术领域的技术人员所理解的含义相同。如无特殊说明，本发明所使用的药品或试剂均按照产品说明书使用或采用所属领域的常规使用方法。现根据说明书附图和具体实施方式对本发明的技术方案作进一步说明。
实施例1
圆斑蝰蛇磷脂酶A2特征多肽的筛选和确定
(1)试剂耗材
试剂：甲酸、乙腈、碘代乙酰胺、胰蛋白酶(sigma)，盐酸胍(VETEC，、二硫苏糖醇(BBI Life Sciences)，其它试剂均为分析纯。
仪器：超纯水仪(Milli_Q)、电子天平(METTLERTOLEDO)、冷冻干燥机(LABCONC)、离心浓缩仪(LABCONC)、高分辨质谱(Thermo Scientific，QEplus)、纳升液相系统(ThermoScientific,EASY-nLC 1000)。
(2)样品处理
样品溶解：圆斑蝰蛇粗毒取适量配置成1μg/μL；
还原烷基化：取50μL(1μg/μL)样品溶液，加入80μL变性缓冲液(称0.606g Tris，8.04mg EDTA，5.73g盐酸胍，加水10mL溶解，用HCl调pH至8.1)及20μL 1M DTT，60℃反应30min；加入40μL 1M IA，避光反应30min；
脱盐：10k超滤膜水洗一次后，加入还原烷基化样品，12000rpm离心，加400μL12000rpm离心，重复3次。
酶解：脱盐后样品加入100μL 25mM NH4HCO3溶解，加2μg胰蛋白酶于37℃过夜酶解，酶解结束后高温灭活并冻干；
复溶：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；
(3)实验条件
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
(4)Proteome Discoverer搜库
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/)中的蛇毒磷脂酶数据库；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。
2特征多肽筛选
质谱结果中酸性磷脂酶A2B亚基(Q7T3T5)和酸性磷脂酶A2 A亚基(Q7T2R1)覆盖率分别是87％和80％。对检测到的特征肽进行筛选，该特征肽需要具有特异性，为圆斑蝰蛇磷脂酶A2特有，长度适宜，不含错且或漏切的酶切位点，不含易发生修饰或降解氨基酸，并且质谱响应较高；最终选择，LIDAKQEAFSFFK作为圆斑蝰蛇磷脂酶A2特征多肽，与数据库Blast序列比对结果(图1)，证明所述特征肽是圆斑蝰蛇磷脂酶A2专有的，可用于磷脂酶A2的种属鉴别。
对圆斑蝰蛇粗毒中找到的特征多肽，通过高分辨质谱对其进行验证，中均能通过Proteome Discoverer2.5匹配到相应肽段，其二级质谱图见图2。
实施例2
三重四级杆质谱法种属鉴别
1.样品制备
供试品溶液：待测样品用25mM碳酸氢铵配制为浓度1μg/μL；取样品溶液100μL，加1μg胰蛋白酶于37℃过夜酶解，酶解结束后高温灭活；12000rpm离心10min，取上清液即得供试品溶液。
空白溶液：取25mM碳酸氢铵100μL，同法制备。
2.实验方法
色谱柱：Waters ACQUITY UPLC BEH C18色谱柱(50mm*2.1mm，1.7μm)；流动相A：0.1％甲酸溶液流动相，B：0.1％甲酸乙腈；按表2进行梯度洗脱；柱温：40℃；进样量：2μL；流速：0.3ml/min。
表2梯度洗脱表
质谱条件：电喷雾离子源(ESI)，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；定性、定量离子对碰撞能量见表3。
表3圆斑蝰蛇磷脂酶A2特征多肽质荷比及质谱条件设置
2.实验结果
取空白溶液、圆斑蝰蛇粗毒及矛头蝮蛇粗毒进行分析，空白溶液及矛头蝮蛇粗毒(图3)在样品出峰位置没有干扰峰出现。圆斑蝰蛇粗毒提取离子色谱图见图4，特征多肽出峰时间为4.4min，质谱响应良好。以上结果表明，该方法可用于圆斑蝰蛇粗毒磷脂酶A2种属鉴别。
分别取10批次含尖吻蝮蛇粗毒、10批次圆斑蝰蛇粗毒、10批次矛头蝮蛇粗毒及10批次白眉蝮蛇粗毒随机取样通过液相色谱-三重四级杆质谱法测进行分析，发现使用本发明所述的特征多肽可以百分百的鉴别出圆斑蝰蛇磷脂酶A2。
以上所述仅说明了本发明的几个实施方式，并不能因此而理解是对本发明专利范围的限制。应当指出，对于本领域的其他人员来说，在不脱离本发明的构思和范围的情况下，还可进行修改替换改进等，这些都属于本发明的保护范围。因此，本发明的专利保护范围应以所描述的根据权利要求为准。
Claims (6)
1.圆斑蝰蛇磷脂酶A2特征多肽，其特征在于，所述特征多肽氨基酸序列为LIDAKQEAFSFFK。
2.一种权利要求1所述圆斑蝰蛇磷脂酶A2特征多肽的应用，其特征在于，所述特征多肽可用于鉴别蛇类磷脂酶A2的种属来源；所述蛇类选自尖吻蝮蛇、圆斑蝰蛇、矛头蝮蛇或白眉蝮蛇。
3.根据权利要求2所述的应用，其特征在于，鉴别采用以下步骤：
（1）取待测样品溶解，进行酶解灭活后作为供试品溶液；
（2）水经过酶解处理，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 772.41→294.18及772.41→342.20作为特征多肽的检测离子对，以确定待测样品中是否含有圆斑蝰蛇磷脂酶A2特征多肽中的特征多肽；
（4）如果检测到权利要求1所述的特征多肽,则证明待测样品中含有磷脂酶A2且来源于圆斑蝰蛇；反之则证明待测样品中不含有来源于圆斑蝰蛇的磷脂酶A2。
4.根据权利要求3所述的应用，其特征在于，步骤（3）所述液相色谱-质谱仪中液相和质谱检测条件中，液相条件为：Waters ACQUITY UPLC BEH C18 色谱柱；流动相A：0.1%甲酸溶液流动相，B：0.1%甲酸乙腈；柱温：40℃；进样量：5 μL；流速：0.3 ml/min；按下表进行梯度洗脱：
所述Waters ACQUITY UPLC BEH C18 色谱柱的尺寸为：柱长50 mm，直径2.1 mm，粒径1.7 µm。
5.根据权利要求3所述的应用，其特征在于，步骤（1）的具体操作为：
①待测样品溶解：待测样品用1%碳酸氢铵配制为浓度1 µg/µL；
②酶解：取步骤①溶解后样品100 µL，加2 µg蛋白酶于37℃过夜酶解，酶解结束后高温灭活；
③离心：最后取步骤②酶解灭活的样品，12000 rpm离心10 min，取上清液即得供试品溶液。
6.根据权利要求5所述的应用，其特征在于，所述的蛋白酶为胰蛋白酶。
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