颜氏大疣蛛蛛毒中多肽和蛋白质的多样性分析

Determination of Peptide and Protein Diversity in the Venom of Macrothele yani

对颜氏大疣蛛Macrothele yani蛛毒所富含的多肽与蛋白质多样性进行探索,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和超高效液相色谱-电喷雾-四极杆-飞行时间质谱技术分离和鉴定颜氏大疣蛛蛛毒中的蛋白质和多肽,并对其相对分子质量分布多样性进行分析。结果显示:粗毒中所含蛋白质的相对分子质量主要分布在35kDa以上。在17~135kDa分离度较佳的共有11条电泳条带,主要集中于40~120kDa附近;在75kDa附近弥散着高丰度的蛋白条带,75kDa以上的蛋白质最丰富。粗毒经色谱分离后得到超过50个色谱峰,经质谱鉴定得到121个物质成分,其中,多肽类物质的相对分子质量呈双峰式分布,21%分布在500~2000 Da,76%分布在3000~5000Da,为粗毒中多肽含量最丰富的部分,且集中于35~60min的保留时间内被洗脱。研究结果表明,颜氏大疣蛛蛛毒中含有较为丰富的多肽和蛋白类物质,这些物质的相对分子质量分布特征与已报道的其他蜘蛛既有相似性又存在具体差异。本文展示了颜氏大疣蛛蛛毒的分子多样性,为后续该毒素的物质基础研究及药用价值开发提供参考。

This study investigated the diversity of peptides and proteins in the venom of Macrothele yani.In this experiment,sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ultra-performance liquid chromatography-electrospray-quadrupole-time of flight mass spectrometry(UPLC-ESI-Q-TOF-MS)were used to separate and identify proteins and peptides in the venom,and the distribution of molecular mass was also analyzed.The results showed that the relative molecular mass of the proteins contained in the crude venom was mainly distributed between 17 kDa and 135 kDa,and there were 11 clearly separated electrophoretic bands with molecular mass ranged from 40 kDa to 120 kDa.Additionally,high-abundance protein bands were dispersed around 75 kDa,and the most abundant bands were found over 75 kDa.Furthermore,more than 50 chromatographic peaks were separated by UPLC and 121 constituents were identified by MS analysis.21%peptides distributed in the range of 500 Da to 2 000 Da while 76%were distributed in the range of 3 000 Da to 5 000 Da showing a bimodal distribution.As the most abundant peptides content in the crude venom of M.yani,the 76%peptides were eluted during the retention time of 35 min to 60 min.The results show that the venom of M.yani contains abundant peptides and proteins.However,this study lays a foundation for the material basic research and drug development of the toxin in the future.