Using a CRISPR/Cas9-based approach, we engineered human primary CD4+ and CD8+ T cells in which a bait protein (LAT, SLP76, VAV1 or ZAP70) was tagged with an affinity Twin-Strep-tag (OST), with the purpose of determining by quantitative mass spectrometry the composition and dynamics of the signalosome assembling around each of these signaling proteins prior to and following T cell activation. Affinity purification of the OST tagged protein was performed using Streptactin beads, from T cells left non-stimulated, or stimulated for 30s, 60s, 120s, or 300s with anti-CD3 and anti-CD28 antibodies. Each AP-MS purification is associated with a corresponding control (purification from non-edited WT CD4+ or CD8+ T cells, cultured and stimulated in the same conditions). The number of replicate biological experiments was n=3 for all time-points, and each sample was analyzed twice by single-run nano LC-MS.