Two‐dimensional gel electrophoresis for proteome projects: The effects of protein hydrophobicity and copy number

作者：Marc Ronald Wilkins, Elisabeth Gasteiger, Jean‐Charles Sanchez, Amos Bairoch, Denis F. Hochstrasser · 发表于：Electrophoresis · 年份：1998 · DOI：10.1002/elps.1150190847 · 被引用次数：201 · 研究领域：Advanced Proteomics Techniques and Applications、Mass Spectrometry Techniques and Applications、Genomics and Phylogenetic Studies

Two-dimensional (2-D) gel electrophoresis is often used in proteome projects to provide a global view of the proteins expressed in any cell or tissue type. Here we have investigated the effects of protein hydrophobicity and cellular protein copy number on a protein's presence or absence on a two-dimensional gel. The average hydropathy values of all known proteins from Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae were calculated, thus defining the range of protein hydrophobicity and hydrophilicity in these organisms. The average hydropathy values were then calculated for a total of 427 proteins from these species, which had been identified elsewhere on 2-D gels. Strikingly, it was seen that no highly hydrophobic proteins, as defined by average hydrophobicity values, have been found to date on 2-D gel separations of whole cell lysates. A clear hydrophobicity cutoff point was seen, above which current 2-D electrophoresis methods appear not to be useful for protein separation. The effect of cellular protein copy number on a protein's presence on a 2-D gel was investigated by means of a graphical model. This model showed how variations in protein loading and copy number per cell interact to determine the quantity of a protein that will be present on a 2-D gel. Considering the current maximum in 2-D gel loading capacity, it was found that 2-D probably can not visualize or produce analytical quantities of proteins present at less than 1000 copies per cell. We con...