Discovering and validating unknown phospho-sites from p38 and HuR protein kinases in vitro by Phosphoproteomic and Bioinformatic tools

We have combined different resins: (a) IMAC (Immobilized Metal Affinity Capture), (b) TiO2 (Titanium dioxide) and (c) SIMAC (Sequential Elution from IMAC) to isolate phosphopeptides from p38 and HuR protein kinases in vitro.Different phosphopeptide MS strategies were carried out by the LTQ ion Trap mass spectrometer (Thermo): (a) Multistage activation (MSA) and (b) Neutral loss MS3 (DDNLMS3).In addition, Molecular Dynamics (MD) bioinformatic simulation has been applied in order to simulate, over a period of time, the effects of the presence of the extra phosphate group (and the associated negative charge) in the overall structure and behaviour of the protein HuR.This study is supported by the Declaration of Helsinki and subsequent ethical guidelines.The combination of these techniques allowed for:(1) The identification of 6 unknown phosphopeptides of these protein kinases. (2) Amino acid site assignments of the phosphate groups from each identified phosphopeptide, including manual validation by inspection of all the spectra. (3) The analyses of the phosphopeptides discovered were carried out in four triplicate experiments to avoid false positives getting high reproducibility in all the isolated phosphopeptides recovered from both protein kinases. (4) Computer simulation using MD techniques allowed us to get functional models of both structure and interactions of the previously mentioned phosphorylated kinases and the differences between their phosphorylated and un-phosphorylated forms.Many research studies are necessary to unfold the whole signalling network (human proteome), which is so important to advance in clinical research, especially in the cases of malignant diseases.As with other MAPK pathways, the p38 signalling cascade involves sequential activation of MAPK kinase kinases (MAP3Ks) and MAPK kinases (MKKs) including MKK3, MKK4, and MKK6, which directly activate p38 through phosphorylation in a cell-type- and stimulus-dependent manner [1,2]. Once activated,