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The pathological importance of tumor necrosis factor (TNF)-alpha in rheumatoid arthritis (RA) is now widely accepted. Ex vivo data from synovial cell cultures suggest that direct cell contact between activated T-cells and macrophages may be an important driver of macrophage TNF-alpha production in the RA joint. However, the ligand/receptor pairs driving this cell contact signal remain obscure. One reason for this is that plasma membrane (PM) proteins are resistant to systematic analysis using traditional proteomic approaches. In this chapter we present a method for the enrichment and resolution of PM proteins from murine T-cell hybridomas as a prelude to identification by tandem mass spectrometry. We used cell surface biotinylation, differential centrifugation and subsequent streptavidin affinity capture, followed by solution phase iso-electric focussing and tandem mass spectrometry to identify 75 PM proteins and make semiquantitative comparisons of resting and activated cells. The method is applicable to a wide variety of cell types.

Type

Journal article

Journal

Methods in molecular medicine

Publication Date

01/2007

Volume

136

Pages

361 - 367

Addresses

Kennedy Institute of Rheumatology Division, Imperial College London, UK.

Keywords

Lymphocytes, T-Lymphocytes, Hybridomas, Cell Membrane, Animals, Mice, Arthritis, Rheumatoid, Tumor Necrosis Factor-alpha, Membrane Proteins, Proteome