关键词: 蛋白质, 多肽, 反相高效液相色谱, 基质辅助激光解吸离子化飞行时间质谱, 家福捕鸟蛛, 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳, 蜘蛛粗毒

Abstract:

Selenocosmia jiafu (S. jiafu) is recently identified as a new species of spider in P. R. China. These medium bodied venomous spiders are distributed mainly in the hilly areas of southwest of China, mostly at Yunnan and Guangxi Provinces. In order to understand the composition of the S. jiafu venom, we performed a preliminary analysis of this venom using reversed-phase high performance liquid chromatography (RP-HPLC), matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The S. jiafu venom was separated by RP-HPLC in an analytical C18 column (phenomenex 100 Å, 250 mm×4.6 mm, 5 μm) equilibrated with solution A (distilled water with 0.1% trifluoroacetic acid), using a gradient from 0% to 50% of solution B (acetonitrile with 0.1% trifluoroacetic acid) over 50 min with a flow rate of 1 mL/min. The isolated venom proteins were treated with in-gel digestion separated by SDS-PAGE and then identified by liquid chromatography-electrospray ionization quadrupole-time of flight mass spectrometry (LC-ESI-QTOF-MS) techniques. The results show that more than 40 fractions eluted were monitored at 215 nm in the RP-HPLC chromatogram of the venom of the spider S. jiafu. Most of the fractions were eluted with retention times of 5-15 min and 25-40 min, corresponding to 5%-15% and 25%-40% acetonitrile, respectively. The venom contains 238 peptides that follow a bimodal distribution, with about 62.5% of the peptides having a relative molecular mass of 3000-4500 and about 33.2% of the peptides having a relative molecular mass of 1000-3000. This distribution model is rather different from those of peptides from other tarantula spider venoms analyzed. To explore the relative molecular mass distribution of the venom proteins, the venom was analyzed by SDS-PAGE using standard protocols. Except for peptides with relative molecular mass lower than 10000, the SDS-PAGE electrophoresis revealed three more obvious bands around 50, 72 and 90 kD respectively. Further MS analysis indicated that there are mainly hemocyanin, potassium ion channel protein, calcium protease and so on. Altogether, this study not only indicated there are many peptides and proteins in the S. jiafu venom, but also provided a basis for further case-by-case investigation of peptide toxins from this venom.

Key words: Selenocosmia jiafu (S. jiafu), matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), peptides, proteins, reversed-phase high performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), spider venom