A robust method for proteomic characterization of mouse cartilage using solubility-based sequential fractionation and two-dimensional gel electrophoresis.
Identification of protein expression differences using two-dimensional electrophoresis (2-DE) and multidimensional liquid chromatography (MDLC)-based proteomics depends critically on reproducibility throughout sample preparation and analysis. This applies particularly where sample fractionation is used to remove high abundance or interfering components to facilitate deeper mining of the proteome. Here we present a procedure for solubility-based cartilage fractionation using sequential extraction with 1 M sodium chloride followed by 4 M guanidinium hydrochloride. We characterized the extracts by 1-D electrophoresis and immunoblotting for individual cellular and matrix components and more globally by 2-DE. In general, NaCl extracts were highly enriched for cellular proteins and GuHCl extracts were predominantly matrix components, with some interesting exceptions. Importantly, we observed high inter-sample reproducibility and strong correlation between targeted and global analysis, indicating that our method can be applied to differential proteomic analysis of normal and pathological cartilage sub-proteomes.
Wilson
Richard
R
Murdoch Childrens Research Institute, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia. richard.wilson@mcri.edu.au
Bateman
John F
JF