蛋白负染方法在蛋白质组学中的应用评价

The Evaluation of Reverse Staining on Proteomics

为评价蛋白质负染方法在蛋白质组学分析中的应用,采用负染和考马斯亮蓝染色两种方法对同一样品的双向电泳胶进行染色,取相对应的8对蛋白点,并进行胶内酶解及MALDI-TOF/TOF分析,比较两种方法与质谱的兼容性。图像分析显示,负染方法展示出的蛋白点更多,但三维峰图不如考染明晰;质谱结果显示,8个负染蛋白点中有7个鉴定结果有效,8个考染蛋白点鉴定结果均有效。因此可以得出以下结论:负染的灵敏度高于考染,与质谱的兼容性良好,适用于建立双向电泳参考图谱的研究;但负染后的胶图不适于进行蛋白点丰度对比分析。

To evaluate the application of reverse staining in proteomic research, the same protein samples were run by two-dimensional electrophoresis, then the gels were dyed by reverse staining and coomassie Brilliant Blue （CBB） staining. The reverse staining and CBB staining gels were compared, eight pairs corresponding protein spots from the two different gels were cut out, digested by trypsin and analyzed by MALDI-TOF/TOF. Image analysis showed that the reverse staining gel could display more protein spots than the CBB staining one. Seven of eight protein spots on reverse staining gel were effectively identified by MALDI-TOF/TOF, and eight protein spots on CBB staining gel. So, we consider that the sensitivity of reverse staining is higher than the CBB staining, and its compatibility with MALDI-TOF/TOF is good. There-fore, the reverse staining method can be used to set up the reference map of 2-DE, but not suitable to use on the comparative of proteins.