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

Progress in the field of proteomics is dependent upon an ability to visuali se close to an entire protein complement via a given array technology. Thes e efforts have previously centred upon two-dimensional gel electrophoresis in association with immobilised pH gradients in the first dimension. Howeve r, limitations in this technology, including the inability to separate hydr ophobic, basic, and low copy number proteins have hindered the analysis of complete proteomes. The challenge is now to overcome these limitations thro ugh access to new technology and improvements in existing methodologies. Pr oteomics can no longer be equated with a single two-dimensional electrophor esis gel. Greater information can be obtained using targeted biological app roaches based upon sample prefractionation into specific cellular compartme nts to determine protein location, while novel immobilised pH gradients spa nning single pH units can be used to display poorly abundant proteins due t o their increased resolving power and loading capacity. In this study, we s how the effectiveness of a combined use of two differential subproteomes (a s defined by relative solubilities, cellular location and narrow-range immo bilised pH gradients) to increase the resolution of proteins contained on t wo-dimensional gels. We also present new results confirming that this metho d is capable of displaying up to a further 45% of a given microbial proteom e. Subproteomics, utilising up to 40 two-dimensional gels per sample will b ecome a powerful tool for near-to-total proteome analysis in the postgenome era. Furthermore, this new approach can direct biological focus towards mo lecules of specific interest within complex cells and thus simplify efforts in discovery-based proteome research.