Abstract
The physicochemical properties of plasma membrane proteins of mammalian cells render them refractory to systematic analysis by two-dimensional electrophoresis. We have therefore used in vivo cell surface labeling with a water-soluble biotinylation reagent, followed by cell lysis and membrane purification, prior to affinity capture of biotinylated proteins. Purified membrane proteins were then separated by solution-phase isoelectric focusing and SDS-PAGE and identified by high-pressure liquid chromatography electrospray/tandem mass spectrometry. Using this approach, we identified 42 plasma membrane proteins from a murine T cell hybridoma and 46 from unfractionated primary murine splenocytes. These included three unexpected proteins; nicastrin, osteoclast inhibitory lectin, and a transmembrane domain-containing hypothetical protein of 11.4 kDa. Following stimulation of murine splenocytes with phorbol ester and calcium ionophore, we observed differences in expression of CD69, major histocompatibility complex class II molecules, the glucocorticoid-induced TNF receptor family-related gene product, and surface immunoglobulin M and D that were subsequently confirmed by Western blot or flow cytometric analysis. This approach offers a generic and powerful strategy for investigating differential expression of surface proteins in many cell types under varying environmental and pathophysiological conditions.
Highlights
The physicochemical properties of plasma membrane proteins of mammalian cells render them refractory to systematic analysis by two-dimensional electrophoresis
Two-color Fluorescence-activated Cell Sorting (FACS)—Cells were washed twice in FACS buffer (10 mM phosphate-buffered saline (PBS), pH 7.2, 2% w/v BSA, 0.05% w/v sodium azide) and incubated in the same buffer supplemented with mouse immunoglobulin (Ig) G (100 g/ml, 1h, 4 °C)
Our strategy was to label with biotin solvent-exposed lysine residues on the surface proteins of intact cells [10] prior to affinity purification of tagged proteins using SA-agarose beads
Summary
Cell Culture—The mouse T cell hybridoma clone 11A2 was derived and propagated in RPMI 1640 containing 10 mM glutamine and 25 mM HEPES (Biowhittaker, Wokingham, UK) further supplemented with: 10% fetal calf serum (Labtech, Ringmer, UK); 10 mM sodium pyruvate (Sigma, Poole, UK); 10 U/ml penicillin/streptomycin (Biowhittaker), and 10 mM -mercaptoethanol (Life Technologies, Inc., Paisley, UK) as previously described [21]. Beads were washed five times in ice-cold RIPA buffer (10 mM Tris-HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% w/v deoxycholate, 1% w/v Triton X-100, 0.1% w/v SDS), rinsed four times in 1% Triton X-100, and eluted (1 h, room temperature) into extraction buffer (8 M urea, 1% w/v Triton X-100, 1% w/v dithiothreitol) for IEF-based separations, or by boiling into 2ϫ SDS-PAGE. Membranes were washed (4 ϫ 15min) in TTBS and incubated (1 h, room temperature) with appropriate horseradish peroxidase (HRP)conjugated secondary antibodies in TTBS-BSA. Two-color Fluorescence-activated Cell Sorting (FACS)—Cells were washed twice in FACS buffer (10 mM PBS, pH 7.2, 2% w/v BSA, 0.05% w/v sodium azide) and incubated in the same buffer supplemented with mouse immunoglobulin (Ig) G (100 g/ml, 1h, 4 °C). Cells were rinsed in PBS and visualized using an UltraView LCI confocal microscopy suite (Perkin Elmer, Beaconsfield, UK)
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