杂色鲍血淋巴双向电泳与质谱分析

Two-dimensional electrophoresis and mass spectrometry analysis of Haliotis diversicolor hemolymph

以杂色鲍(Haliotisdiversicolor)血淋巴为材料进行了双向电泳研究。首先,确定了以丙酮沉淀法和RC DC蛋白定量试剂盒为基础的蛋白制备与定量方法;其次,对样品前处理方法进行了摸索,通过500 r/min的低速离心方法分离了血细胞和血浆组分,通过35 000 r/min超速离心2 h除掉了部分高丰度的血蓝蛋白,降低了高分子量区域的血蓝蛋白背景;进一步使用18 cm pH 3-10非线性胶条和标准化的电泳程序,对不同上样量的电泳银染结果进行了比较,获得了良好的双向电泳图谱;最终选取肌动蛋白、原肌球蛋白、胶原蛋白和血蓝蛋白4个蛋白点成功进行了质谱鉴定。

Hemolymph of abalone was used as the material for the two-dimensional electrophoresis research. At first, protein preparation and quantification method based on the acetone precipitation and the RC DC Protein Assay Kit was successfully set up. Then pre-treatment methods with 500 r/min low-speed centrifugation and 35000 r/rain ultracentrifugation were used for the separation of hemocyte and blood plasma or reducing the high molecular weight region＇s background. Furthermore, non-linear strips （18 cm, pH 3-10） and standardized electrophoresis protocols were established to evaluate different sample volumes with silver stain. Good two-dimensional electrophoresis maps were finally acquired, and four protein spots in the map were successfully identified by mass spectrometry, which are actin, tropomyosin, collagen and hemocyanin.