Extended Data Fig. 1: Workflow for the analysis of T cell phosphoproteome as a function of TCR ligand affinity. | Nature Immunology

Extended Data Fig. 1: Workflow for the analysis of T cell phosphoproteome as a function of TCR ligand affinity.

From: Kinetic proofreading through the multi-step activation of the ZAP70 kinase underlies early T cell ligand discrimination

Extended Data Fig. 1

(a) Experimental workflow to quantify phosphorylations occurring in the 10 min following TCR stimulation with N4, T4 or G4 under equal TCR occupancy conditions as described in Fig. 1a. OT-1 T cells were stimulated with N4, T4 or G4 for 30, 120, 300, or 600 seconds before lysis, protein digestion and MS analysis. Control samples were harvested without stimulation (0 s). (b) Schematics of the multiplexing strategy employed to compare phosphorylation intensities across a large set of samples. Peptides from different stimulatory condition were labelled with isobaric tags (TMT) enabling multiplexed MS analysis. Six independent experiments consisting of three replicates with N4 and T4 stimulations (N4-T4) and three replicates with N4 and G4 stimulations (N4-G4) were conducted and analyzed to generate the phosphoproteomic dataset. (c) Strategy for phospho-peptide enrichment and MS analysis (see the Methods section for more details).

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