Total phosphorylation in the absence of LAT.

(A) Phosphoproteomics workflow used in this study starts with labeling Jurkat cell lines in different SILAC media in two time series (TS1 and TS2) as indicated, each composed of three sets of differentially labeled cells. In each TS, cell are activated for the indicated time points and the total protein lysates are equally mixed prior to its digestion by trypsin. The resulted peptides are submitted to strong cation exchange chromatography (SCX) and titanium oxide (TiO2) affinity enrichment prior to liquid chromatography and mass spectrometry (LC-MS/MS) analysis. The data is then analyzed (see Methods) and the two time series are normalized using the common time point (0.5 min) so as to obtain a curve representing fold change versus activation time points. (B) Pie chart showing the distribution of the total 11,454 unique phosphorylation sites as phospho (p)-Serine (pS), p-Threonine (pT) and p-Tyrosine (pY). (C) Bar chart presenting the quantified (Qt) and significant change (SChg.) phosphorylated residues as the percent of the total detected sites in CL20 Jurkat cell lines (LAT+/+) or JCaM2.5 (LAT−/−). (D, E) Scatter plot of change in phosphorylation between consecutive time points (Red: 0 to 0.5 min, Green: 0.5 to 5 min, Blue: 0.5 to 10 min, Black: 10 to 20 min) versus the intensity of phosphopeptides - identified in both intact and perturbed cell lines (common peptides) - over each time interval. The grey zone delimits the 95% confidence interval (at ±0.42). (F) Bar chart quantifies the common phosphopeptides classified as significant in (E) as the percent of the total common peptides over the indicated time intervals.