<p>Two cell populations of human Jurkat T cell clones (J14-76-11 and J14) were incubated with RPMI 1640 medium containing normal or heavy isotope labeled arginine and lysine amino acids, physically differentiating the two proteomes by a shift in molecular weights. Each cell population was then pre-incubated with OKT3 and OKT4 antibodies for 10 minutes at 4°C and then crosslinked with anti-IgG at 37°C for the times indicated. After cell lysis, light and heavy cell lysates were combined at an equal protein concentration ratio for each timepoint. Proteins were then reduced, alkylated, and trypsin-digested into peptides. Peptides were desalted by Sep-Pak cartridges, enriched by phosphotyrosine peptide immunoprecipitation and Fe<sup>3+</sup> IMAC, and then subjected to reversed-phase LC-MS/MS analysis. MS shifts introduced by heavy isotope labeling allow for differentiation between light and heavy peptide counterparts in MS spectra. Selected ion chromatogram (SIC) peak areas of light and heavy isotope labeled phosphopeptides were calculated for relative quantification of peptide abundance. Individual SIC peak areas were normalized to the SIC peak area of the copurified synthetic peptide LIEDAEpYTAK in the same timepoint. A label-free heatmap was generated based on peptide abundance for a certain peptide in SLP-76 reconstituted Jurkat cells through a time course of receptor stimulation and SILAC ratio heatmaps were generated based on the ratio of abundance between light (SLP-76 reconstituted) and heavy (SLP-76 deficient) peptide counterparts for each timepoint (SLP-76 deficient in relative to SLP-76 reconstituted).</p
