CN116462749B - 促肝细胞生长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检测及应用领域,特别涉及促肝细胞生长素特征多肽组及其应用。一组促肝细胞生长素特征多肽,氨基酸序列为VGGQAGAHGAEALER及TYFPHFNLSHGSDQVK,来源于血红蛋白α亚基。使用上述特征多肽表征待测样品中促肝细胞生长素的种属来源。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,选择定性定量离子对,对待测样品中促肝细胞生长素的种属来源进行检测;该方法简便快捷,定量准确,填补了促肝细胞生长素的质量标准的空白,提高了质量控制水平。
Description
技术领域
本发明涉及化学分析检测及应用领域,特别涉及促肝细胞生长素特征多肽组及其应用。
背景技术
促肝细胞生长素是从健康的乳猪或未哺乳新生牛新鲜肝脏中提取纯化制备而成具有生物活性的小分子多肽类活性物质,其生产通过研磨、匀浆、反复冻融及加热变性等步骤,是一组相对复杂的混合物,并非单一物质。具有刺激正常肝细胞合成并促进肝细胞再生的生物功能,在临床上广泛应用于治疗急性肝炎、慢性肝炎、重型肝炎及肝硬化的治疗。
对于不同种属来源的促肝细胞生长素,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,目前标准中均采用双缩脲鉴别反应,专属性差,无法对种属来源进行有效检验。
发明内容
为克服现有技术的缺陷,本发明提供了促肝细胞生长素特征多肽组,以及其在表征待测样品中促肝细胞生长素种属来源的技术应用,该技术方法简便快捷,定量准确,填补了促肝细胞生长素质量标准的空白,提高了质量控制水平。
为实现上述发明目的,本发明采用以下技术方案:
促肝细胞生长素特征多肽组,其技术特征为,所述特征多肽组由特征多肽1和特征多肽2组成,其中特征肽1的氨基酸序列为TYFPHFNLSHGSDQVK,特征肽2的氨基酸序列为VGGQAGAHGAEALER。促肝细胞生长素特征多肽组,特征肽1氨基酸序列为TYFPHFNLSHGSDQVK,特征肽2氨基酸序列为VGGQAGAHGAEALER。
进一步地,上述促肝细胞生长素特征多肽组的应用,使用上述特征多肽对待测样品中促肝细胞生长素的种属来源进行检测,采用以下步骤:
(1)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;
(2)取水经过胰蛋白酶酶解预处理,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938.95→733.36及938.95→684.83作为特征肽1的定性定量离子对;以质荷比m/z 双电荷711.86→1081.53及711.86→1010.49作为特征肽2的定性定量离子对;
(4)在以步骤(3)所述的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TYFPHFDLSHGSAQVK及VGGHAAEYGAEALER,证明待测样品来源于猪;反之,则为非猪来源;
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:取待测样品用1%碳酸氢铵溶液配置成1 mg/ml溶液;取200 μL,加1 mg/mL 胰蛋白酶溶液5 μL,37℃酶解过夜,100℃灭活5 min,放冷至室温,1200 rpm离心10 min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:
液相条件:Waters Acquity Premier Peptide CSH C18,色谱柱 100 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→3 min,流动相B 3%;2→8 min,流动相B 3%→60%;8.1→10min,流动相B 90%;10.1→12min,流动相B 3%。
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:5.5 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;特征肽1碰撞能量CE为45 V,特征肽2碰撞能量CE为50 V。
上述方法中,所述待测样品为促肝细胞生长素。对于特征肽的筛选,除了质谱响应较高之外,稳定且普遍存在于待测样品之中也十分重要。对于促肝细胞生长素,其生产工艺存在加热变性等步骤,是一组相对复杂的混合物,蛋白组学分析中,检测到大量随机断裂位点。不同企业工艺不同,随之产生的断裂位点也会有差异,产生的肽段序列差异也会较大。因此,选择一组稳定存在于各企业且质谱响应较高的特征肽,十分困难。本次实验通过对几家企业十几批促肝细胞生长素原液及注射液分析,筛选后得到的促肝细胞生长素特征多肽组,该组特征肽质谱响应好,专属性强,且稳定存在于不同企业不同批次样品之中。对该组特征肽进行检测,除了能对原料的种属来源进行检测外,也能对反应企业内部的工艺稳定性。
(1)经大量实验研究及蛋白质数据库比对,本发明寻找到了促肝细胞生长素的特征多肽段(TYFPHFDLSHGSAQVK及VGGHAAEYGAEALER),由于已知马牛羊等动物的血红蛋白氨基酸序列中均不含该段氨基酸序列,因此可用于检测待测样品中促肝细胞生长素的种属来源。
(2)本发明公开的定性定量方法简便快捷,定量准确,填补了促肝细胞生长素质量标准的空自,可以大大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。
附图说明
图1 猪牛羊马血红蛋白α亚基序列比对图;
图2 促肝细胞生长素特征多肽1二级质谱图;
图3 促肝细胞生长素特征多肽2二级质谱图;
图4 空白溶液猪源特征肽提取离子色谱图;
图5 牛血红蛋白特征肽提取离子色谱图;
图6 促肝细胞生长素特征肽提取离子色谱图。
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)、促肝细胞生长素(广东隆赋药业及广州一品红药业)、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)1%碳酸氢铵溶液:称取0.1g碳酸氢铵,加50 mL水溶解,即得。
(3)二硫苏糖醇(DTT)溶液:称取15.42 mg 二硫苏糖醇,加500 μL水溶解,即得。
(4)碘乙酰胺(IA)溶液(临用新制):称取18.5 mg碘乙酰胺,加500 μL水溶解,即得。
(5)1 mg/mL胰蛋白酶溶液(临用新制):称取胰蛋白酶10 mg,加10 mL水溶解,即得。
实施例
促肝细胞生长素特征多肽的筛选和确定
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 3-30 K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:赛默飞Acclaim PepMap®100 C18纳升色谱柱:75 μm × 15 cm(3μm,100Å)和100μm × 2 cm(5μm,100 Å)。
流动相A:2% 乙腈的0.1% 甲酸水溶液;流动相B: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2.0kV,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
Time(min) | Flow(nL/min) | B(%) |
0 | 300 | 3 |
5 | 300 | 8 |
85 | 300 | 28 |
102 | 300 | 38 |
110 | 300 | 95 |
120 | 300 | 95 |
3 数据采集
样品溶解:分别取广州一品红制剂及原液各三批、广东隆赋制剂及原液各三批适量配置成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 1MDTT,60℃反应45 min;加入30 µL 1M IA,避光反应45 min;
脱盐:C18固相萃取小柱先用2mL 乙腈活化并用2mL 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2mL 0.1%TFA水溶液脱盐;最后2mL 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1µg 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,12000 rpm离心10 min,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,蛋白组分析结果表明,广州一品红及广东隆赋两家企业,匹配到最多PSMs的蛋白均为血红蛋白α亚基和β亚基,覆盖率在80%以上。考虑到选择种属鉴别时需要选择动物中特有且稳定存在的肽段,因此,选择血红蛋白中的肽段进行种属鉴别。结合肽段筛选原则和质谱响应,对VGGQAGAHGAEALER和TYFPHFNLSHGSDQVK两特征多肽组进行种属特异性分析,通过对猪牛羊马血红蛋白α亚基序列比对(图1),发现两条肽是猪血红蛋白特有的。通过uniprot blast分析表明,TYFPHFNLSHGSDQVK肽段除了猪外,也在两种猴科中存在;VGGQAGAHGAEALER除猪外,也在草原西瑞中存在,但是其他动物中均没有发现同时存在两种特征多肽。因此该两条特征肽联合使用可用于猪源促肝细胞生长素的种属鉴别。
实施例
多反应监测(MRM)定性分析样品中的促肝细胞生长素。
1 仪器设备
三重四级杆液质联用仪(AB SCIEX 6500+)
2 色谱质谱条件
液相条件:Waters Acquity Premier Peptide CSH C18,色谱柱 100 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→3 min,流动相B 3%;2→8 min,流动相B 3%→60%;8.1→10min,流动相B 90%;10.1→12 min,流动相B 3%。
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:5.5 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;特征肽1碰撞能量CE为45 V,特征肽2碰撞能量CE为50 V。
3 溶液制备
供试品溶液:取待测样品5 mg,置10 mL量瓶中,用1%碳酸氢铵溶液溶解并定容;量取200 μL, 加1 mg/mL 胰蛋白酶溶液5 μL,37℃酶解过夜,100℃灭活5 min,放冷至室温,1200 rpm离心10 min得上清液。
空白溶液:精密量取1%碳酸氢铵溶液200 μL,同法操作,作为空白溶液。
4 测定法
取上述待测溶液各2 μL,按照2项下色谱质谱条件检测。
5专属性考察
取两家企业各三批次原液及三批次制剂及牛血红蛋白进样分析,空白溶液(图4)在样品出峰位置没有干扰峰出现。样品猪源特征肽提取离子色谱图见图6,特征肽1(猪)出峰时间为5.1 min,特征肽2(猪)出峰时间为3.9 min,两家企业原液及制剂均能检测到相应色谱峰。在牛血红蛋白样品中(图5),特征肽1 提取离子色谱图没有明显色谱峰,特征肽2提取离子色谱图中的两个色谱峰与样品保留时间有明显差异。以上结果表明,该方法可用于猪源促肝细胞生长素种属鉴别,且两家企业原液及制剂均为猪源。
取10批含有促肝细胞生长素的样品、10批次牛血红蛋白样品、10批次羊血红蛋白样品、10批次羊血红蛋白样品和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促肝细胞生长素的样品。
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 (4)
1.一种促肝细胞生长素特征多肽组的应用,其技术特征为,所述特征多肽组由特征多肽1和特征多肽2组成,其中特征肽1的氨基酸序列为TYFPHFNLSHGSDQVK,特征肽2的氨基酸序列为VGGQAGAHGAEALER;使用上述特征多肽组对待测样品中促肝细胞生长素的种属来源进行检测;
具体采用以下步骤:
(1)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;
(2)取水经过胰蛋白酶酶解预处理,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938.95→733.36及938.95→684.83作为特征肽1的定性定量离子对;以质荷比m/z 双电荷711.86→1081.53及711.86→1010.49作为特征肽2的定性定量离子对;
(4)在以步骤(3)所述的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TYFPHFDLSHGSAQVK及VGGHAAEYGAEALER,证明待测样品来源于猪;反之,则为非猪来源。
2.根据权利要求1所述的应用,其技术特征为,步骤(1)所述经过胰蛋白酶酶解预处理的具体操作为:
①取待测样品用1%碳酸氢铵溶液配置成1 mg/ml溶液;
②取200 μL,加1 mg/mL 胰蛋白酶溶液5 μL,37℃酶解过夜;
③最后取步骤②酶解后的样品100℃灭活5 min,放冷至室温,1200 rpm离心10 min得上清液。
3.根据权利要求1所述的应用,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 Premier Peptide CSH C18,色谱柱 100 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→3 min,流动相B 3%;2→8 min,流动相B 3%→60%;8.1→10 min,流动相B90%;10.1→12 min,流动相B 3%。
4.根据权利要求1所述的应用,其特征在于,所述液相色谱-质谱仪中质谱条件如下: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;特征肽1碰撞能量CE为45 V,特征肽2碰撞能量CE为50 V。
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