tailieunhanh - Báo cáo y học: "Discovering and validating unknown phosphosites from p38 and HuR protein kinases in vitro by Phosphoproteomic and Bioinformatic tools"

LTuyển tập các báo cáo nghiên cứu về y học được đăng trên tạp chí y học Wertheim cung cấp cho các bạn kiến thức về ngành y đề tài: Discovering and validating unknown phosphosites from p38 and HuR protein kinases in vitro by Phosphoproteomic and Bioinformatic tools. | López et al. Journal of Clinical Bioinformatics 2011 1 16 http content 1 1 16 JOURNAL OF CLINICAL BIOINFORMATICS RESEARCH Open Access Discovering and validating unknown phosphosites from p38 and HuR protein kinases in vitro by Phosphoproteomic and Bioinformatic tools Elena Lopez1 5 Isabel Lopez2 Julia Sequí3 and Antonio Ferreira4 Abstract Background The mitogen activated protein kinase MAPK pathways are known to be deregulated in many human malignancies. Phosphopeptide identification of protein-kinases and site determination are major challenges in biomedical mass spectrometry MS . P38 and HuR protein kinases have been reported extensively in the general principles of signalling pathways modulated by phosphorylation mainly by molecular biology and western blotting techniques. Thus although it has been demonstrated they are phosphorylated in different stress stimuli conditions the phosphopeptides and specific amino acids in which the phosphate groups are located in those protein kinases have not been shown completely. Methods 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. Results 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 .