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 biol ogical systems. Proteins in the molecular mass region of 20-120 kDa are wel l investigated and described. However, proteins with masses below 20 kDa ar e the least investigated as they are rarely seen on 2D-PAGE due to fast mig rations in the electric field and lack of staining efficiency. This paper d escribes a technique that enriches proteins in the lower mass region using solid-phase extraction. The purification step is carried out using C-18 fun ctionalised "restricted access" affinity chromatography whereby simultaneou s trace enrichment and sample clean up is achieved. In this study expressio n patterns of TGF-beta stimulated and non-stimulated fibroblasts were compa red after the solid-phase fractionation procedure. An increased expression pattern was obtained whereby 400 protein spots could be detected by image a nalysis in the <20-kDa region. Out of these, specific regulations of 14 spo ts were found by quantitative image analysis and spots of interest were ide ntified with MALDI TOP-MS. The regulated and identified proteins were trios ephosphate isomerase, cofilin and heat shock 27-kDa protein. (C) 2001 Elsev ier Science B.V. All rights reserved.