高效毛细管电泳指纹图谱法检测全蝎酶解液中蛋白类成分(>10 kDa)的实验研究
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摘要
建立了用高效毛细管电泳指纹图谱法来检测全蝎酶解液中蛋白类成分(>10k Da)的分析方法。采用未涂层弹性熔融石英毛细管柱(93 cm×0.75μm),有效柱长为79 cm;二极管阵列检测器;50 mbar压力进样10s;运行电压为-30 kV;柱温为20℃;运行时间为60 min;考察不同检测波长、缓冲溶液种类、pH、浓度等对色谱峰的影响,并对10批样品做了相似度评价。在缓冲溶液浓度为0.2 mol·L~(-1)、pH=5.0硼酸缓冲溶液时响应值较高,谱图效果最好;检测波长为220 nm时出峰较多、吸收好,初步建立了以25个峰为共有峰的指纹图谱。结论:25个峰的高效毛细管电泳指纹图谱共有模式可以作为全蝎酶解液中蛋白类成分(>10 kDa)的鉴别方法。
A method of protein detection of scorpion enzymolysis solution(>10 k Da) by HPCE was established.Elastic-uncoated-fused silica capillary column(93 cm×0.75 μm) with 79 cm effective column length was used as well as diode array detector, the sampling time at 50 mbar was 10 s, the operating voltage was 30 k V, the column temperature was 20 ℃, the operation time was 60 min.The effect of detection wavelength, buffer solution type, pH and concentration on chromatograms was investigated, meanwhile the similarity evaluation of 10 batches of samples was carried out. The results showed that, when 0.2 mol·L~(-1) borate with pH=5.0 was used as the buffer solution, the response value was higher, and the spectrum was the best. When the detection wavelength was 220 nm, the peak kinds was multiple and the UV absorbance was stronger, and the fingerprint spectrum of 25 peaks was established preliminarily. At last, it's pointed out that common model of HPCE chromatographic fingerprints of the 25 peaks can be used to detect the proteins of scorpion enzymolysis solution(>10 kDa).
A method of protein detection of scorpion enzymolysis solution(>10 k Da) by HPCE was established.Elastic-uncoated-fused silica capillary column(93 cm×0.75 μm) with 79 cm effective column length was used as well as diode array detector, the sampling time at 50 mbar was 10 s, the operating voltage was 30 k V, the column temperature was 20 ℃, the operation time was 60 min.The effect of detection wavelength, buffer solution type, pH and concentration on chromatograms was investigated, meanwhile the similarity evaluation of 10 batches of samples was carried out. The results showed that, when 0.2 mol·L~(-1) borate with pH=5.0 was used as the buffer solution, the response value was higher, and the spectrum was the best. When the detection wavelength was 220 nm, the peak kinds was multiple and the UV absorbance was stronger, and the fingerprint spectrum of 25 peaks was established preliminarily. At last, it's pointed out that common model of HPCE chromatographic fingerprints of the 25 peaks can be used to detect the proteins of scorpion enzymolysis solution(>10 kDa).
引文
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[15]徐毅励,陈媛梅,郑彩霞.高效毛细管电泳法研究重瓣榆叶梅花中蛋白质的动态变化[J].中国农业大学学报,2016(05):76-82.
[16]汪勇,高培峰,赵新颖,屈锋.毛细管电泳法表征多肽及糖蛋白的稳定性[J].色谱,2013(06):543-549.
[17]侯连兵,陈振德,陈志良,许重远,杨西晓.三七及其混淆品蛋白多肽高效毛细管电泳法鉴别[J].中草药,2000(11):61-62.
[18]李敏,武晓林,张影,赵权.高效毛细管电泳法同时测定桦褐孔菌中7种核苷类成分的含量[J].中国药学杂志,2017(02):152-156.
[19]曾艳萍,刘训红,李俊松.沙苑子的HPCE指纹图谱鉴别[J].现代中药研究与实践,2007(04):18-20.
[2]冯成强,张文生,崔光红,黄璐琦.半夏及其混淆品水半夏的蛋白高效毛细管电泳法鉴别[A].中国中医科学院.中医药发展与人类健康——庆祝中国中医研究院成立50周年论文集(下册)[C].中国中医科学院:,2005:2.
[3]刘婷,姜金斗,刘宁.毛细管电泳检测奶粉中添加的大豆分离蛋白[J].分析科学学报,2008(01):37-41.
[4]石燕.荷斯坦牛乳蛋白毛细管电泳指纹图谱的建立及应用[D].东北农业大学,2010.
[5]陶然.中药刺五加和麻黄的毛细管电泳指纹图谱研究[D].重庆大学,2006.
[6]李乐,陈志涛,夏之宁,杨丰庆,张新娜.毛细管电泳法在测定蛋白质或多肽的理化特性中的应用[J].化学通报,2014(01):12-18.
[7]杜旭.高效毛细管电泳在中医药领域的最新进展和应用研究[J].天津药学,2011(05):33-37.
[8]霍玉荣.超高效液相色谱法在药物分析中的应用进展[J].首都食品与医药,2016(10):25-26.
[9]孔毅,吴如金,吴梧桐.高效毛细管电泳及其在蛋白质、多肽分析中的应用[J].药学进展,2000(04):204-208.
[10]贾萍.指纹图谱与中药质量控制[J].中国药业,2004(02):79-80.
[11]陈志良,陈振德,侯连兵,许重远,刘建武,杨西晓.枸杞子蛋白多肽的高效毛细管电泳法鉴别[J].中国药学杂志,2001(03):48-50.
[12]孙毓庆,孙国祥,金郁.毛细管电泳指纹图谱及毛细管电泳-质谱联用在中药质量控制中的作用[J].色谱,2008(02):160-165.
[13]王贤纯,范春明,唐新科,陈平,梁宋平.牛血清白蛋白胰蛋白酶酶解产物的色谱-质谱联用分析及其三种数据库搜寻鉴定方法的比较[J].中国生物化学与分子生物学报,2004(03):393-398.
[14]陈振德,许重远,庄志铨,侯连兵.穿山甲及其炮制品蛋白多肽高效毛细管电泳法鉴定[J].广东药学院学报,2000(04):302-304.
[15]徐毅励,陈媛梅,郑彩霞.高效毛细管电泳法研究重瓣榆叶梅花中蛋白质的动态变化[J].中国农业大学学报,2016(05):76-82.
[16]汪勇,高培峰,赵新颖,屈锋.毛细管电泳法表征多肽及糖蛋白的稳定性[J].色谱,2013(06):543-549.
[17]侯连兵,陈振德,陈志良,许重远,杨西晓.三七及其混淆品蛋白多肽高效毛细管电泳法鉴别[J].中草药,2000(11):61-62.
[18]李敏,武晓林,张影,赵权.高效毛细管电泳法同时测定桦褐孔菌中7种核苷类成分的含量[J].中国药学杂志,2017(02):152-156.
[19]曾艳萍,刘训红,李俊松.沙苑子的HPCE指纹图谱鉴别[J].现代中药研究与实践,2007(04):18-20.