Zooming-in on the proteome: Very narrow-range immobilised pH gradients reveal more protein species and isoforms

Two-dimensional gel electrophoresis (2-DE) enables separation of complex mi xtures of proteins on a single polyacrylamide gel according to isoelectric point, molecular weight, solubility, and relative abundance. For this reaso n, 2-DE together with mass spectrometry (MS) has become a key technology in proteome analysis. The introduction of immobilised pH gradients (IPGs) for isoelectric focusing of proteins affords improved reproducibility and perm its full-scale proteome analyses to be undertaken. Whilst broad-range IPGs are useful for investigating simple proteomes (e.g. Mycoplasma genitalium) it is becoming clear that additional resolving power is needed for separati ng the more complex proteomes of eukaryotic organisms. The use of narrow-ra nge and very narrow-range IPGs provides the means with which to dissect a c omplex proteome. We have compared very narrow-range IPGs (3.5-4.5L, 4-5L, 4 .5-5.5L, 5-6L, and 5.5-6.7L) with broad- (3-10NL) and narrow-range IPGs (4- 7L and 6-9L) for the visualisation of the human heart proteome. The superio r ability of very narrow-range IPGs to separate different protein species a nd isoforms, compared with 3-10NL and 4-7L 2-D gels is demonstrated. The re sults are supported by MS identifications which further show that reduction of the number of comigrating protein species results in less ambiguous and more reliable database search results.