Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, United States.
Anal Chem. 2011 Mar 1;83(5):1784-90. doi: 10.1021/ac103148k. Epub 2011 Feb 10.
Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) is a fundamental analytical technique for proteomic research, and SDS-capillary gel electrophoresis (CGE) is its miniaturized version. Compared to conventional slab-gel electrophoresis, SDS-CGE has many advantages such as increased separation efficiency, reduced separation time, and automated operation. SDS-CGE is not widely accepted in proteomic research primarily due to the difficulties in identifying the well-resolved proteins. MALDI-TOF-MS is an outstanding platform for protein identifications. Coupling the two would solve the problem but is extremely challenging because the MS detector has no access to the SDS-CGE-resolved proteins and the SDS interferes with MS detection. In this work we introduce an approach to address these issues. We discover that poly(tetrafluoroethylene) (PTFE) membranes are excellent materials for collecting SDS-CGE-separated proteins. We demonstrate that we can wash off the SDS bound to the collected proteins and identify these proteins on-membrane with MALDI-TOF-MS. We also show that we can immunoblot and Coomassie-stain the proteins collected on these membranes.
十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）是蛋白质组学研究中的一种基本分析技术，而 SDS-毛细管凝胶电泳（SDS-CGE）则是其微型化版本。与传统的平板凝胶电泳相比，SDS-CGE 具有许多优点，例如提高了分离效率、缩短了分离时间和实现了自动化操作。然而，SDS-CGE 在蛋白质组学研究中并未得到广泛应用，主要是因为难以识别得到良好分离的蛋白质。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）是一种出色的蛋白质鉴定平台。将两者结合可以解决这个问题，但极具挑战性，因为 MS 检测器无法接触到 SDS-CGE 分离的蛋白质，而且 SDS 会干扰 MS 检测。在这项工作中，我们介绍了一种解决这些问题的方法。我们发现聚四氟乙烯（PTFE）膜是收集 SDS-CGE 分离蛋白质的绝佳材料。我们证明可以洗掉与收集的蛋白质结合的 SDS，并在膜上使用 MALDI-TOF-MS 对这些蛋白质进行鉴定。我们还展示了可以对这些膜上收集的蛋白质进行免疫印迹和考马斯亮蓝染色。
