Top-down proteomics : 2D gels are an integral part of the process

Two-dimensional gel electrophoresis (2DE) remains widely regarded as a gold-standard for proteomic analyses. Nonetheless, issues with the method have been routinely noted in the ‘review’ literature, although there has been little to substantiate these claims and many do not seem plausible when the 2DE technique is considered from the perspective of its underlying chemistry and that of proteins. As (or perhaps because) gel-based proteomics is a ‘mature’ technology, factors contributing to possible reductions in performance are known and thus it is possible to better optimize ongoing analyses by targeted refinement of the technique; in contrast, issues with other approaches that have been popularised over the last decade are only now being more widely recognised. Here I briefly review efforts from my group to quantitatively improve every stage of 2DE analysis, from sample preparation and protein extraction, to in-gel protein detection approaches for the enhancement of total proteome coverage. As the overall objective is to provide optimal analyses of (patho)physiological mechanisms, we have applied this refined 2DE protocol in proteomic investigations of human preterm labour, spinal cord injury, and a number of other conditions/samples relevant to both basic and clinical sciences.