Subproteomics based upon protein cellular location and relative solubilities in conjunction with composite two-dimensional electrophoresis gels

Abstract

Progress in the field of proteomics is dependent upon an ability to visualise close to an entire protein complement via a given array technology. These efforts have previously centred upon two-dimensional gel electrophoresis in association with immobilised pH gradients in the first dimension. However, limitations in this technology, including the inability to separate hydrophobic, basic, and low copy number proteins have hindered the analysis of complete proteomes. The challenge is now to overcome these limitations through access to new technology and improvements in existing methodologies. Proteomics can no longer be equated with a single two-dimensional electrophoresis gel. Greater information can be obtained using targeted biological approaches based upon sample prefractionation into specific cellular compartments to determine protein location, while novel immobilised pH gradients spanning single pH units can be used to display poorly abundant proteins due to their increased resolving power and loading capacity. In this study, we show the effectiveness of a combined use of two differential subproteomes (as defined by relative solubilities, cellular location and narrow-range immobilised pH gradients) to increase the resolution of proteins contained on two-dimensional gels. We also present new results confirming that this method is capable of displaying up to a further 45% of a given microbial proteome. Subproteomics, utilising up to 40 two-dimensional gels per sample will become a powerful tool for near-to-total proteome analysis in the postgenome era. Furthermore, this new approach can direct biological focus towards molecules of specific interest within complex cells and thus simplify efforts in discovery-based proteome research.

Strategies for the enrichment and identification of basic proteins in proteome projects

“…For example, highly hydrophobic membrane proteins are biologically designed to be insoluble in solution, therefore they remain nearly impossible to solubilize for electrophoretic purposes. Similarly, many low abundance proteins are present on 2-D gels, but they cannot be visualized due to the overwhelming presence of abundant 'housekeeping' proteins, and may only become visible when separated in conjunction with narrow range IPGs [9][10][11].…”

“…9.0) are rarely separated using 2-DE. In recent times, novel IPGs have become available for separating basic proteins within the pH ranges 6-11 [14][15][16][17], 7-10, 8-11 [18] and 9-12 [1,9]. Alkaline proteins are technically challenging for 2-DE for several reasons.…”

Pharmacoproteomics in drug development

“…This improves MS/MS performance and significantly increases the number of proteins identified and quantified. Use of 13 C as the heavy ICAT isotope (rather than deuterium) improves coelution of both heavy and light isotopes in reversed-phase chromatography and increases accuracy of comparative quantification. Additionally, different chemistries and approaches to differentially tagged protein samples have been developed, 44,45 and stable isotope incorporation methods for cells grown in culture have been devised.…”

“…To that end, novel sample preparation steps aimed at increasing protein recovery and resolution have been reported. [11][12][13] Despite their shortcomings, 2D gel-based experiments remain an exceptional approach for assessing differential protein expression, particularly in studies with pharmacological considerations.…”

Two-dimensional gel electrophoresis in bacterial proteomics

“…These were far more robust and uniform in nature, and are now widely used due to their excellent reproducibility. Both linear and non-linear pH gradients, which span a number of different pH ranges, are now commercially available (Bjellqvist et al, 1982;Cordwell et al, 2000;Görg et al, 2000;Patton, 2002).…”