UFLC-MS/MS法分析蜈蚣中3种多肽成分以及在蜈蚣鉴别中的应用

  目的  建立超高效液相色谱-质谱联用(UFLC-MS/MS)法分析蜈蚣中3种酶解多肽成分TD1(LEEDLERSEERL)、TD2(EEKDKALQNAEGEVAAL)、TD3(MILPTGASSF), 比较不同品种蜈蚣中肽类成分的差异, 寻找蜈蚣特异性多肽, 用于蜈蚣的鉴别。  方法  3种肽类成分的UFLC-MS/MS法, 色谱柱为XSelect HSS T₃(4.6 mm×150 mm, 3.5 μm); 流动相为乙腈-0.1%甲酸溶液，梯度洗脱。采用三重四极杆质谱检测器, 电喷雾离子化(ESI), 正离子模式下多反应监测(MRM)进行信号采集。样品采用正交试验方法, 对蛋白提取方法进行优化, 最终确定提取方法为蛋白裂解液超声处理60 min。得到的蜈蚣蛋白提取溶液经胃蛋白酶酶解并脱盐后, 注入液质联用仪分析。测定了20批蜈蚣及8批其他动物药中3种肽类成分, 并对结果进行比较。  结果  在地龙、僵蚕、水蛭、土鳖虫和全蝎中未检出TD2和TD3, TD1与TD2在少棘巨蜈蚣与哈氏蜈蚣、墨江蜈蚣中存在较大差异, TD3在少棘巨蜈蚣与黑头蜈蚣、多棘蜈蚣中有较大差异。  结论  该方法可用于区分少棘巨蜈蚣与地龙、僵蚕、水蛭、土鳖虫和全蝎, 同时通过比较3种肽段的差异, 区分药典品种少棘巨蜈蚣和4种常见非药典品种蜈蚣, 为蜈蚣的质量评价和基础研究提供参考。 

Analysis of Three Peptide Components in Centipede by UFLC-MS/MS and Its Application in Identification of Centipede

  OBJECTIVE  To analyze three enzymatic peptide components TD1 (LEEDLERSEERL), TD2 (EEKDKALQNAEGEVAAL) and TD3 (MILPTGASSF) in centipedes by UFLC-MS/MS method for comparing the differences of peptide components in different species of centipedes and identification of centipedes.  METHODS  UFLC-MS/MS of the three peptide components was performed on an XSelect HSS T₃ (4.6 mm×150 mm, 3.5 μm) column and the mobile phase was acetonitrile -0.1% formic acid solution with gradient elution. A triple quadrupole mass spectrometer detector with electrospray ionization (ESI) and multiple reaction monitoring (MRM) in positive ion mode were used for signal acquisition. The samples were subjected to orthogonal experimental methods to optimize the protein extraction method, and the final extraction method was determined by sonicating the protein lysate for 60 min to obtain the centipede protein extraction solution, which was enzymatic hydrolyzed and desalted before injecting. Three peptide components were determined in 20 batches of centipedes and 8 batches of other animal medicines and the results were compared.  RESULTS  TD2 and TD3 were not detected in earthworm, stiff silkworm, leech, ground beetle and scorpion. TD1 and TD2 were more different in S. subspinipes mutilans L.Koch and S. subspinipes dehaani Brandt and S. mojiangica Zhang et chi, and TD3 was more different in S. subspinipes mutilans L.Koch and S. negrocapitis Zhang et Wang and S. subspinipes multidens Newport.  CONCLUSION  This method can be used to distinguish the S. subspinipes mutilans L. Koch from those of earthworm, stiff silkworm, leech, ground beetle and scorpion. By comparing the differences of three peptides, Scolopendra subspinipes mutilans L.Koch can be distinguished with other four common non-pharmacopoeial species, providing a reference for quality evaluation and basic research of centipedes.