Towards higher resolution: Two-dimensional Electrophoresis ofSaccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients

Abstract

The rising number of proteome projects leads to new challenges for two-dimensional electrophoresis with immobilized pH gradients and different applications of this technique. Not only wide pH gradients such as 4-12 or 3-12 (Görg et al., Electrophoresis 1999, 20, 712-717) which can give an overview of the total protein expressions of cells are in demand but also overlapping narrow immobilized pH gradients are to be used for more specialized and detailed research and micropreparative separations. The advantage of overlapping narrow pH gradients is the gain in higher resolution by stretching the protein pattern in the first dimension. This simplifies computer-aided image analysis and protein identification (e.g., by mass spectrometry). In this study the protein patterns of yeast cells in pH gradients 4-5, 4.5-5.5, 5-6, 5.5-6.7 and 6-9 are presented and compared to the pH 4-7 and 3-10 gradients. This combination allowed us to reveal a total of 2286 yeast protein spots compared to 755 protein spots in the pH 3-10 gradient.

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

“…Some of these preparative methods do not yield high-resolution separations and/or do not produce sufficiently small sample volumes for direct application to analytical 2-D gels at high protein loads. A conceptually attractive alternative to sample prefractionation is direct analysis of samples using multiple narrow pH range IPG gels in parallel [16][17][18]. The main benefits of narrow IPG gradients are that the total number of protein spots per pH unit can be increased substantially due to higher spatial resolution of different protein spots.…”

“…The use of sample cup loading rather than gel rehydration loading could potentially reduce artifacts caused by precipitation of proteins with pIs outside narrow pH range IPG gels, since these proteins only contact the gel at the sample loading site [17]. However, sample cup loading methods can be problematic.…”

Methods for fractionation, separation and profiling of proteins and peptides

“…They estimate that the increase in resolution allowed for the detection of proteins present at only about 300 copies per cell. Wildgruber et al [46] compared the collective use of IPG strips with pH ranges of 4-5, 5-6, and 5.5-6.7 against gels run with IPG strips with pH ranges 3-10 and 4-7. They found that they were able to detect 2.3 and 1.6 times more protein spots using the narrow-range gels than with the IPG 3-10 and 4-7 gels, respectively.…”

“…Overlapping spots were only counted once. Reprinted from [46], with permission. dimensional separation technique for the analysis of proteome mixtures.…”

Comparison of lysis methods and preparation protocols for one- and two-dimensional electrophoresis of Aspergillus oryzae intracellular proteins

“…Alternatively, a proteomics approach would be useful for functional genomics studies. While 2-DE has been widely used to study the proteome of a number of industrially relevant organisms [6], including both bacteria [7] and yeast [8], only very few reports on 2-DE of filamentous fungi are available, and these are typically strains of medical [9] verses industrial importance.…”