不同上样方式对蛋白质组双向电泳图谱质量的影响比较

蛋白质组技术难点之一是如何获取尽可能多的细胞或组织的蛋白信息.双向电泳蛋白斑点数目直接反映了实验蛋白质组信息的完整性.除样品制备外,蛋白上样方式对双向电泳图谱的质量和完整性有直接的影响.实验论文从以下3个方面考察不同的蛋白上样方式对双向电泳图谱的影响;即：水化上样与杯上样；一次上样与重复上样；以及酸性端加样与碱性端上样.实验结果发现;蛋白上样量较大时,杯上样方式的图谱斑点数目较水化上样方式明显增多；样品蛋白浓度较高时,稀释多次上样明显优于一次性浓缩蛋白上样；蛋白裂解液(MCF7乳腺癌细胞)在酸性端加样对偏碱性蛋白的分离未发现明显优势.相反,在等电聚焦伏小时(Vh) 足够的前提下,碱性端加样对偏碱性端蛋白反而有利,表现为斑点数目较多,而且等电点方向拖尾减轻.实验结果对提高双向电泳的质量以及相关蛋白质组信息的完整性提供了有益的技术参考.

Map Quality of Two-dimensional Gel Electrophoresis Affected by Different Sample-loading Methods

The number and resolution of isolated protein spots are two critical factors for two-dimensional gel electrophoresis (2-DE)-based proteome study. In this paper, three different sample-loading methods were evaluated in terms of these two factors: cup sample loading vs. rehydration loading; once-in-all sample (cup) loading vs. multiple loading and, anodic side sample loading vs. cathodic side loading. MCF-7 and HEP-2 cell lysis suspensions were used for 2-DE separation. The spot number was improved by 49.5% for cup loading mode as compared with rehydration loading. Also, 54% more protein spots were observed from multiple loading than once-in-all loading mode. Moreover, cup loading mode could increase spot volume, which facilitates down-stream protein identification by mass spectrometry. In addition, sample loading position may affect protein separation: anodic side loading seems favor acidic-like protein isolation while cathodic side loading seems better for basic-like protein.