Supplementary Figure 2: Whole-cell proteomics of CD4+ T cells during TCR activation identifies upregulation of TCR-associated proteins and pathways.

a, Pairwise comparisons of protein abundance measured from WCP mass spectrometry experiments in CD4+ T cells rested or restimulated for 4 h with CD3 + CD28 antibody coated beads (3:1 cell:bead ratio). For label free quantification (exp 1), log2 normalized iBAQ values were used to represent protein abundance at rest or restimulation. For SILAC quantification (exp 2 and 3), log2 normalized “heavy” intensity was used for the resting cell protein abundance and “light” intensity was used for restimulated cell protein abundance. Correlation coefficients were calculated for all-by-all pairwise comparisons of three experiments, using the Pearson’s method. b, MetaCore (portal.genego.com) pathway enrichment within the significantly upregulated proteins identified in the WCP mass spectrometry experiments in CD4+ T cells unstimulated or stimulated, as described in (a). Analysis was performed on proteins exhibiting log2 fold change > 0 and p-value < 0.05, based on a two-tailed Students-t test. Enriched pathways were identified by FDR, based on a q-value calculation, performed by the MetaCore program. c, Histograms of log2 fold changes in protein abundance from WCP mass spectrometry experiments in CD4 + T cells unstimulated or stimulated, as described in (a). Log2 fold changes are compared for 1 h stimulation (gray) and 4 h stimulation (black). d, MS/MS counts from the label-free quantified WCP mass spectrometry experiments in CD4 + T cells rested or restimulated, as described in (a), for 1 or 4 h. MS/MS counts are used as a measure of protein abundance for proteins known to be induced upon CD4 + T cell activation. Rest (0 h) and 4 h data are reproduced from main Fig. 2d to provide for comparison with 1 h data.