Session: Session 4

PRM assay of Diacylglycerol Kinase Kappa

Fragile X syndrome (FXS) is the most common inherited cause of autism and intellectual disabilities worldwide. It is caused by defaults in a gene called Fragile X Messenger Ribonucleoprotein 1 (FMR1). A Fmr1-KO mouse model which recapitulates the symptoms is characterized by inhibition of diacylglycerol kinase kappa (DGKk).  Furthermore DGKk-KO mouse model induces symptoms and inversely, DGKk re-expression in Fmr1-KO corrects them. All these results produced at RNA level make it possible to envisage a treatment by controlling the level of DGKk expression. Unfortunately, the detection and quantification of DGKk in the various genetic backgrounds is impossible, as no good specific antibody has yet been produced.

Mouse DGKk was overexpressed in Hela cells and global proteomic analysis after IP identified 35 tryptic peptides. Three peptides were selected for assay and heavy/light forms were synthesized for standard curves and matrix effect analysis. Absolute quantification of DGKk was achieved by MS ratio of target peptides and their SIL counterpart. Brain tissue samples were extracted with RIPA buffer and a short gel migration was used to prefractionate DGKk in a migration zone between 100 and 200 kDa before in-gel tryptic digestion. An Exploris 480 with FAIMS module was set up with a PRM method and a short nanoLC gradient. Data were processed with Skyline.

DGKk is specifically detected in WT thanks to the three peptides and four y-ions per peptide (y4 to y7). Various brain regions were sampled, but DGKk is only identified in hypothalamus extracts of WT mice (Hypothalamus has the higher level of specific RNA). Conversely no signal is obtained with Dgkκ-KO mice. The PRM assay of DGKk provides a powerful tool to quantify the protein in 15 min. These preliminary data validate the DGKk mouse and the assay will allow to study the effect of different gene therapy approaches for the restoration of DGKk expression in the hypothalamus of Dgkκ-KO mice.