Department of Ecology and Biology, Tuscia University, Viterbo, Italy.
Nat Protoc. 2011 Dec 8;7(1):36-44. doi: 10.1038/nprot.2011.427.
Proteomic analysis of red cells is compromised by the presence of high-abundance proteins (hemoglobin and carbonic anhydrase-1), which completely obscure low-abundance species. The depletion method presented here involves performing native gel electrophoresis in a polyacrylamide gel tube using a modified electroelution cell. The electrophoretic run is interrupted intermittently to allow the recovery of at least three different liquid fractions, which can be analyzed by both native PAGE and 2D isoelectric focusing SDS-PAGE, or by shotgun mass spectrometry analysis after trypsin in-solution protein digestion. This low-cost, reproducible technique can be used to process large amounts of sample, and it increases the likelihood of detecting low-abundance proteins, thereby resulting in greater proteome coverage. The separation procedure takes approximately 6-7 h.
红细胞的蛋白质组分析受到高丰度蛋白质（血红蛋白和碳酸酐酶-1）的干扰，这些蛋白质完全掩盖了低丰度的物种。这里介绍的耗尽方法涉及在聚丙酰胺凝胶管中进行天然电泳，使用改良的电洗脱池。电泳运行间歇性中断，以允许至少三种不同的液体部分恢复，这些部分可以通过天然 PAGE 和 2D 等电聚焦 SDS-PAGE 进行分析，也可以在溶液中胰蛋白酶消化蛋白质后通过鸟枪法质谱分析进行分析。这种低成本、可重复的技术可用于处理大量样品，并增加了检测低丰度蛋白质的可能性，从而提高了蛋白质组的覆盖率。分离过程大约需要 6-7 小时。
