Restricted access chromatographic sample preparation of low mass proteins expressed in human fibroblast cells for proteomics analysis

Two-dimensional electrophoresis and modern image analysis systems have made it possible to study protein expression and regulation of proteins in biological systems. Proteins in the molecular mass region of 20-120 kDa are well investigated and described. However, proteins with masses below 20 kDa are the least investigated as they are rarely seen on 2D-PAGE due to fast migrations in the electric field and lack of staining efficiency. This paper describes a technique that enriches proteins in the lower mass region using solid-phase extraction. The purification step is carried out using C-18 functionalised "restricted access" affinity chromatography whereby simultaneous trace enrichment and sample clean up is achieved. In this study expression patterns of TGF-beta stimulated and non-stimulated fibroblasts were compared after the solid-phase fractionation procedure. An increased expression pattern was obtained whereby 400 protein spots could be detected by image analysis in the <20-kDa region. Out of these, specific regulations of 14 spots were found by quantitative image analysis and spots of interest were identified with MALDI TOP-MS. The regulated and identified proteins were triosephosphate isomerase, cofilin and heat shock 27-kDa protein. (C) 2001 Elsevier Science B.V. All rights reserved.