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CD4+ T helper cells are the key player for control and regulation of adaptive immune responses. In a healthy immune system they protect the body against a variety of pathogens but CD4+ T cells are also involved in pathogenesis and perpetuation of diverse autoimmune diseases like chronic rheumatic inflammation. Consequently, analysis of T cell activation has the potential to assist the development T cell-directed therapies and diagnostic approaches. Membrane proteins are of specific interest because they represent 60% of known drug targets and have also the potential to serve as diagnostic markers.
The membrane proteomes of a well characterized set of freshly isolated and CD3/CD28-stimulated human CD4+ T cells were analyzed by high resolution 2D-IEF-SDS-PAGE. Membrane proteins were enriched by differential centrifugation and following Triton-X114 extraction. Reproducibility of this isolation method for membrane proteins and 2D PAGE was proven by comparison of proteomes of resting and activated CD4+ T cell samples derived from individual human donors. Using differential centrifugation and detergent extraction protein spots were displayed in proteomes that were not detectable or underrepresented in 2D gels of whole cellular lysates without any fractionation.
Furthermore, for quality control of the procedure a selection of well known surface antigens was quantitatively measured by FACS analysis prior to electrophoresis. These antigens were stained by large 2D-PAGE western blot of the separated T cell proteins identified by mass spectrometry. They were assigned to the corresponding 2D gel maps for quantitative spot analysis. Recovery as well as expression ratios of surface antigens between resting and activated T cells were compared.