Analysis of the interactome of CD6 in CRISPR/CAS9 modified mouse CD4+ T cells

Using a CRISPR/Cas9-based approach we engineer primary CD4+ T cells in which the CD6 protein 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 the tagged protein prior to and following T cell activation. Affinity purification of the OST tagged protein was performed using Streptactin beads, from T cells that were non-stimulated, or stimulated for 30s or 120s with anti-CD3 and anti-CD4 antibodies, or 300s with pervanadate. Each AP-MS purification is associated with a corresponding control (purification from non edited WT CD4+ T cells, cultured and stimulated in the same conditions). The number of replicate biological experiments was n=5 (cells non-stimulated or stimulated for 30s with anti-CD3/CD4), n=2 (cells stimulated for120s with antiCD3/CD4), or n=3 (cells stimulated 300s with pervanadate), and each sample was analyzed in duplicate or triplicate by LC-MS, resulting in 78 raw files.