Characterization of signal transduction by P2X7 in CD4+ T cells by a proteomic approach

ATP released from CD4+helper T cells upon TCR stimulation contributes to the activation of MAPKsignalling through purinergic P2X receptors. It has been shown that Tregs produce lower amounts ofATP than conventional CD4+ T cells, although the gene encoding P2X7 receptor is comprised inTregs signature genes. Activation of purinergic P2X7 receptor by ATP mediates T regs conversion to pro-inflammatory T cells, thus promoting autoimmunity. In this work we identified and characterized the phosphoproteome triggered by BzATP as P2X7 agonist both in stimulated and non-stimulated Tregs employing a SILAC-based proteomic approach in combination with protocols for the enrichment of phosphorylated proteins and high-resolution mass spectrometry. C57BL/6 wild-type (WT) and p2rx7 - /- Tregs labelled with different isotopes (R0K0 or R10K8, respectively) in SILAC media were stimulated with BzATP. After Tregs stimulation, cells were lysed and combined prior to enzymatic digestion. Proteolytic digests were fractionated by HILIC and further enriched on a Titansphere Phos-TiO resin. The phosphopeptides were subsequently separated by HPLC coupled online to an LTQ-Orbitrap velos. The mass spectrometer was operated in positive ion mode in data-dependent acquisition mode to alternate between a full scan (m/z 350-2000) in the Orbitrap and subsequent CID MS/MS in the linear ion trap of the 20 most intense peaks from full scan. For identification and quantification of phosphopeptides, MaxQuant 1.3.0.5 was used with the mouse UniProt database and Andromeda. Search parameters allowed for 2 missed tryptic cleavages, a mass tolerance of 6 ppm in MS mode and 20 ppm in CID MS/MS mode, a static modification (carbamidomethylation) on cysteine, and up to 5 total dynamic modifications [(N-acetylation (protein), oxidization (Met) and phosphorylation (Ser, Thr, and Tyr)]. To achieve highly reliable identifications, the following criteria were used: maximal peptide FDR of 0.01 and minimal peptide length of 6.