Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble and large proteins

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Abstract
The number of unique protein species in proteomes from a single mammalian cell type is not well defined but is likely to be at least 10 000–20 000. Since standard‐size two‐dimensional gels typically resolve only about 1500 to 3000 spots, they merely analyze a small portion of these proteomes. In addition, all insoluble proteins and typically proteins >100 kDa are seldom resolved on two‐dimensional (2‐D) gels. The current study demonstrates the feasibility of an overall strategy for more comprehensive quantitative comparisons of complex proteomes derived from physiological fluids or mammalian cell extracts. A key feature of this approach is to prefractionate samples into a few well‐resolved fractions based on the proteins' isoelectric points (pIs) using microscale solution isoelectric focusing. These fractions are then separated on narrow pH range two‐dimensional gels approximately ±0.1 pH unit wider than the prefractionated pool. When this prefractionation approach is applied to complex mammalian proteomes, it improves resolution and spot recovery at high protein loads compared with use of parallel narrow pH range gels without prefractionation. The minimal cross‐contamination between fractions allows quantitative comparisons in contrast to most alternative prefractionation methods. In addition, complementary data can be obtained by parallel analysis of the solubilized fraction on high‐resolution large‐pore‐gradient one‐dimensional gels followed by mass spectrometric identification to analyze proteins between 100 and ∼500 kDa. Similarly, insoluble proteins can be analyzed using large‐pore gels for large proteins and 10–12% one‐dimensional sodium dodecyl sulfate (SDS) gels for smaller proteins. Together, these strategies should permit more reliable quantitative comparisons of complex mammalian proteomes where detection of at least 10 000 protein spots is needed in order to analyze the majority of the unique protein species.