Comprehensive analysis of B-Raf proteins interactome by mass spectrometry.

BONFIGLIO J.J.; MACCARRONE G.; REWERTS, C.; HOLSBOER, F.; ARZT, E.; TURCK, C.; SILBERSTEIN, S.

Bremen - Alemania

Congreso; 18th International Mass Spectrometry Conference.; 2009

B-Raf is a protein kinase member of the Raf family that encodes serine-threonine kinases central to the activation of the mitogen- activated protein kinases/extracellular-signal-regulated kinases 1/2 (MAPKs/ERK1/2). The MAPKs signal transduction pathway couples intracellular responses to extracellular signals through cell surface receptors. In neurons, the activation of ERK1/2 is involved in cellular processes essential for cell survival and takes place through the B-Raf-MEK1/2-ERK1/2 cascade, since B-Raf is the main Raf family member in the nervous system. The aim of our work is to identify B- Raf-associated proteins using immunoaffinity chromatography combined with mass spectrometry analysis. B-Raf partners identified in this work may allow identification of key components playing a role in the regulation of B-Raf-MEK1/2-ERK1/2 signaling pathway in neurons. Method Mouse hippocampal cell line HT22 has been used for the identification of the associated proteins with B-Raf. For this purpose, we performed non-denaturing immunoprecipitations (IPs) in cell lysates from starved cells (no ERK 1/2 activation) using an anti-B-Raf polyclonal antibody.  The immunoprecipitated proteins were separated by 1D-SDS/PAGE, and the obtained gel slides were subjected to in-gel tryptic digestion. The resulting proteolytic peptides were analyzed by LC-MALDI-MS/MS. Proteins were identified by searching the MS spectra against a mouse database with the support of the Mascot algorithm.      Preliminary Data A pre-clearing step using a commercial rabbit anti-mouse polyclonal antibody was included in the workflow for the sample preparation to prevent any non-specific protein binding to the anti-B-Raf rabbit polyclonal antibody used in the immunoprecipitation. The B-Raf interacting proteins isolated by immunoprecipitation have been analysed by mono dimensional gel electrophoresis, the protein separation  resulted in a limited number of blue-Coomassie stained gel bands.  In order to achieve a depth analysis of the proteins interactome, the entire SDS-gel lane was analyzed following the shotgun mass spectrometry approach. For the identification of low abundant proteins in the B-Raf complex, the peptides resulted from the protelytic digestion have been separated by liquid chromatography before being analysed by MALDI-MS/MS. A large number of proteins (65%) has been identified by more than 2 peptide hits based on Mascot probability score. Some of the identified proteins have been validated by western blot analysis. The functional interactivities between the identified proteins have been investigated in silico by interrogating the public literature with the software Pathway Studio.    Novel Aspects Comprehensive analysis of the B-Raf-associated proteins in HT22 cells with no active MAPK pathway.