CN116675761B - 绒促性素特征多肽组及其应用 - Google Patents
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Abstract
本发明涉及化学分析定性检测技术领域,尤其涉及一种绒促性素特征多肽组及其应用。所述特征多肽组由特征多肽1和特征多肽2组成,具体的氨基酸序列为:特征多肽1氨基酸序列为VLQGVLPALPQVVCNYR,特征多肽2氨基酸序列DHPLTCDDPR。该特征多肽组可用于检测待测样品中绒促性素特征多肽种属来源,检测准确度高,填补了绒促性素质量标准的空白,可以显著提高绒促性素的质量控制水平,确保绒促性素产品临床用药的有效性和安全性。
Description
技术领域
本发明涉及化学分析定性检测技术领域,尤其涉及绒促性素特征多肽组及其应用。
背景技术
绒促性素(HCG)为孕妇尿中提取的绒毛膜促性腺激素,是人胎盘滋养层分泌的一种促性腺激素,绒促性素的化学结构由碳水化合物链和多肽链组成,是一种水溶性糖蛋白,由α和β两个含有糖侧链的亚基以非共价键结合而成,相对分子质量约为30,000~60,000。
药典及注册标准中均对绒促性素蛋白药物来源进行了规定,但质量标准中并未进行种属来源控制。对于不同种属来源的酶类蛋白药物,其多肽序列会有差异,药物药效也会有不同,明确种属来源有利于风险防控及原材料生产溯源。
特征肽检测技术是用质谱测定物种中特有的肽段从对动物源成分进行区分鉴别,其专属性强,精密度高。特征肽检测方法开发涉及肽段筛选(活性成分筛选、差异位点鉴别、酶解条件优化、肽段筛选)、液相条件优化(色谱柱及流动相选择,梯度优化),质谱条件优化(MRM 离子对选择及碰撞能量优化)。筛选具有专属性、稳定且质谱响应较好的特征肽,并建立一种分析方法是比较复杂的过程。
发明内容
为解决上述技术问题,本发明的发明目的是提供绒促性素特征多肽组,所述特征多肽组能够在表征样品中绒促性素的种属来源中发挥重要作用,填补了绒促性素质量标准的空白。
为实现上述发明目的,本发明采用以下技术方案:
所述特征多肽组由特征多肽1和特征多肽2组成;其中特征多肽1的氨基酸序列为VLQGVLPALPQVVCNYR,特征多肽2氨基酸序列DHPLTCDDPR。
一种上述绒促性素特征多肽组的应用,所述特征多肽组用于检测绒促性素的种属来源。
进一步的,上述应用方法采用以下步骤:
(1)取待测样品溶解,进行还原烷基化、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,以质荷比双电荷m/z963.54→1035.50及963.54→398.24作为特征多肽1的检测离子对,以质荷比双电荷m/z 613.27→272.17及613.27→253.09,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,并获得特征多肽1和特征多肽2的含量;
(4)如果同时检测到特征多肽1和特征多肽2,则证明待测样品来源于人;反之,则为非人来源。
具体定性离子对、定量离子对、碰撞能量及去簇电压见下表:
优选地,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(50 mm*2.1 mm,1.7μm);流动相A:0.1%甲酸溶液流动相,B:0.1%甲酸乙腈;按下表进行梯度洗脱;柱温:40℃;进样量:2 μL;流速:0.3 ml/min;
优选地,步骤(2)的具体操作为:
待测样品溶解:待测样品用25 mM碳酸氢铵配制为浓度1 µg/µL;
②还原烷基化:取200µL步骤①待测样品溶液,5µL 1M DTT,60℃反应45min;加入10µL 1M IA,避光反应45 min;
③酶解:取步骤②还原烷基化后样品,加入200 µL 25 mM碳酸氢氨,加4µg 胰蛋白酶于37℃过夜酶解,酶解结束后高温灭活;
④离心:最后取步骤③酶解后样品12000 rpm离心10 min,取上清液即得待测样品。
优选地,所述的DTT溶液和IA溶液为临用新配溶液。
与现有技术相比,本发明具有以下有益效果:由于已知猪牛羊马等常见家畜和动物的氨基酸序列中不含有本发明所述的绒促性素特征多肽,更不会同时含有本发明所述的特征多肽1和特征多肽2,因此,该特征多肽组可用于检测待测样品中绒促性素特征多肽种属来源,检测准确度高,填补了绒促性素质量标准的空白,可以显著提高绒促性素的质量控制水平,确保绒促性素产品临床用药的有效性和安全性。
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为绒促性素α、β3及β7亚基的蛋白组分析结果;
图2为实施例1中绒促性素特征多肽VLQGVLPALPQVVCNYR二级质谱图;
图3为实施例1中绒促性素特征多肽DHPLTCDDPR二级质谱图;
图4为实施例2中空白溶液特征多肽提取离子色谱图;
图5为实施例2中KC211202批次绒促性素特征多肽提取离子色谱图;
图6为实施例2中KC220401批次绒促性素特征多肽提取离子色谱图;
图7为实施例2中KC220503批次绒促性素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
绒促性素特征多肽的筛选和确定
(1)试剂耗材
试剂:甲酸、乙腈、胰蛋白酶(sigma,批号SLBS8956),盐酸胍(VETEC,批号WXBC4261V)、二硫苏糖醇(BBI Life Sciences,批号D911BA0011)、碘代乙酰胺(BBI LifeSciences,批号B326BA1943),Sep-pak C18固相萃取小柱(Waters,批号009836286A),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)缓冲液配制
蛋白变性缓冲液:称量0.606 g Tris,8.04 mg EDTA,5.73 g盐酸胍,加水10 mL溶解,HCl调至pH8.1。
1 M DTT溶液:称量154.2 mg DTT溶于1 mL液质水后配成1 M二硫苏糖醇(DTT)溶液(现用现配);
1 M IA溶液:称量185 mg IA溶于1 mL液质水以配置1 M碘乙酰胺(IA)溶液(现用现配);
25 mM碳酸氢铵溶液:称量79.06 mg NH4HCO3溶于40 mL液质水配成25 mM碳酸氢铵溶液。
(3)样品处理
样品溶解:样品取适量加25 mM碳酸氢铵配置成1 µg/µL;
还原烷基化:取50 µL(1 µg/µL)样品溶液,加入75 µL变性缓冲液及15 µL 1 MDTT,60℃反应45 min;加入30 µL 1 M IA,避光反应45 min;
脱盐:C18固相萃取小柱先用2 mL 乙腈活化并用2 mL 0.1%TFA水溶液平衡;取还原烷基化结束的样品重复上样三次,并用2 mL 0.1%TFA水溶液脱盐;最后2 mL 0.1%TFA 80%ACN溶液洗脱,并将洗脱液使用离心浓缩仪转干;
酶解:转干脱盐后样品加入100 µL 25 mM NH4HCO3溶解,加1 µg 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500 µL复溶并混匀,12000 rpm离心10 min,取上清于进样瓶中进行液质联用分析。
(4)色谱条件
色谱柱为赛默飞Acclaim PepMap®100 C18纳升色谱柱:75 μm×15 cm(3 μm,100Å)和100 μm × 2 cm(5 μm,100 Å)。
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2 μL。采用EASY-nLC 1000纳升液相系统分离。纳升分离泵流速为300 nL/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2.0 kV,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
(5)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/)中的人蛋白数据库;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10 ppm,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(+57.021 Da);可变修饰选为甲硫氨酸氧化(+15.995 Da);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
HCG结构中包括α、β两个亚基,α亚基与LH、FSH、TSH近似,尤其是与LH有较大的免疫交叉反应,β链为其独有。蛋白组分析结果表明,绒促性素α、β3及β7亚基均能检测到,覆盖率分别是29%,66%,61%(图1)。因此我们选择β链进行种属鉴别分析。结合肽段筛选原则及理论酶切肽段(表1)和质谱响应,对VLQGVLPALPQVVCNYR及DHPLTCDDPR进行种属特异性分析,通过uniprot blast分析,猪牛羊马等常见家畜和动物中不存在,因此该两条特征多肽可用于人绒促性素的种属鉴别。
表1 绒促性素理论酶解肽段
3 特征多肽验证
对绒促性素原料中找到特征多肽,通过高分辨质谱对其进行验证,三批原料(KC211202批次、KC220401批次、KC220503批次)中均能通过Proteome Discoverer2.5匹配到相应肽段,其二级质谱图见图2-图3。
实施例2
三重四级杆质谱法种属鉴别
样品制备
供试品溶液:待测样品加25mM碳酸氢氨配制为浓度1 µg/µL;取200µL样品溶液,5µL 1M DTT,60℃反应45 min;加入10 µL 1M IA,避光反应45 min;加入200 µL 25 mM碳酸氢氨,加4 µg 胰蛋白酶于37℃过夜酶解,酶解结束后高温灭活;12000 rpm离心10 min,取上清液即得待测样品。
空白溶液:取200 µL 25 mM碳酸氢氨溶液同法操作。
实验方法
色谱柱: Waters ACQUITY UPLC BEH C18 色谱柱(50 mm*2.1 mm,1.7 μm);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:2 μL;流速:0.3 ml/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:5.5 kV;碰撞室出口电压:10 V;入口电压(EP)10 V;定性、定量离子对碰撞能量见表3:
表3 绒促性素特征多肽质荷比及质谱条件设置
2.实验结果
取空白溶液及三批次(KC211202批次、KC220401批次、KC220503批次)原料进样分析,空白溶液(图4)在样品出峰位置没有干扰峰出现。3批次绒促性素提取离子色谱图见图5-图6,特征多肽1出峰时间为3.6 min,特征多肽2出峰时间为6.7min,3批次绒促性素均能检测到特征多肽1和特征多肽2的相应色谱峰相应色谱峰。以上结果表明,该方法可用于绒促性素种属鉴别。
3.结论
本发明结合蛋白质组和高分辨质谱分析方法对绒促性素进行特征多肽的筛选与确证,样品通过酶解后进行高分辨质谱分析,并通过搜库软件分析:对于绒促性素,我们筛选到VLQGVLPALPQVVCNYR和DHPLTCDDPR各两条特征多肽,与猪牛羊马相比,均是人特有的,因此可联合用于人绒促性素的种属鉴别。之后通过建立了液相色谱-三重四级杆质谱法,分别对三批次绒促性素原料(KC211202批次、KC220401批次、KC220503批次)进行分析,结果表明该方法能用于绒促性素的种属鉴别,上述原料均为人源。
分别取10批次含人绒促性素样品、10批次含猪绒促性素样品、10批次含牛绒促性素样品、10批次含羊绒促性素样品、10批次含马绒促性素样品,将上述50批样品混合放置,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1和特征多肽2可以百分百的鉴别出人源绒促性素。
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 (4)
1.一种绒促性素特征多肽组的应用,其特征在于,所述绒促性素特征多肽组用于检测绒促性素的种属来源;所述特征多肽组由特征多肽1和特征多肽2组成;其中,特征多肽1的氨基酸序列为VLQGVLPALPQVVCNYR,特征多肽2氨基酸序列DHPLTCDDPR;所述应用采用以下步骤:
(1)取待测样品溶解,进行还原烷基化、酶解后作为供试品溶液;
(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,以质荷比双电荷m/z 963.54→1035.50及963.54→398.24作为特征多肽1的检测离子对,以质荷比双电荷m/z 613.27→272.17及613.27→253.09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,并获得特征多肽1和特征多肽2的含量;
(4)如果同时检测到特征多肽1和特征多肽2,则证明待测样品来源于人;反之,则为非人来源。
2.根据权利要求1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;按下表进行梯度洗脱;柱温:40℃;进样量:2 μL;流速:0.3 ml/min;
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。
3.根据权利要求1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:待测样品加25 mM碳酸氢氨配制为浓度1 µg/µL;
②还原烷基化:取200 µL步骤①待测样品溶液,5 µL 1M DTT,60℃反应45 min;加入10µL 1M IA,避光反应45 min;
③酶解:取步骤②还原烷基化后样品,加入200 µL 25 mM碳酸氢氨,加4 µg 胰蛋白酶于37℃过夜酶解,酶解结束后高温灭活;
④离心:最后取步骤③酶解后样品12000 rpm离心10 min,取上清液即得供试品溶液。
4.根据权利要求3所述的应用,其特征在于,所述的DTT溶液和IA溶液为临用新配溶液。
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