Sample Extraction Techniques for Enhanced Proteomic Analysis of Rat Brain Tissue

High resolution two dimensional electrophoresis (2DE) remains the core technology for resolving complex protein mixtures, prior to mass spectrometry characterization. Due to the complexity of brain tissue, optimising protein separation techniques is fundamental to unravel the function of brain proteins and their role in disease. So, brain tissue is a very good model to improve resolution due to either hydrophobic then basic characteristic of proteins. It has been described that for wide pH 3–10 gradients, the use of HED (Hydroxyethyl disulfide) could also be advantageous; although not needed for reduction of streaking, it has been result in an improved reproducibility of the basic part of the resulting 2-D maps. In this work it has been applied HED to perform rat brain two dimensional electrophoresis. Analytical loads in combination with anodic cup application or paper bridge application has been used to compare methods to resolve brain extract. Image analysis indicate that paper bridge loading combined with HED give very sharp image and a strong reduction of streaking and improved separation and focusing of spots according to literature; statistical analysis derived from image analysis shows a loss of spots ( about 20% ) compared with anodic cup loading. Therefore the latter method coupled to HED is the best choice for semi-quantitative analysis of brain tissue.