Background:
Many anti-cancer drugs induce apoptosis. We have performed a quantitative proteome analyses of Sorafenib-induced cell death in human neuroblastoma cells using stable isotope labelling by amino acids in cell culture (SILAC) (Bull et al., J. Proteome Res., 2012, 11, 1609-1620). Sorafenib is a multikinase inhibitor and in clinical use against different types of cancer.
Mitochondria are known as the powerhouses of the cell. Furthermore, mitochondrial outer membrane permeabilisation (MOMP) is an event that is considered to be the point of no return during apoptosis. The balance of anti- and pro-apoptotic members of Bcl-2 family controls this process. If the point of no return is reached, it results in the diffusion of several proteins into the cytosol. Best known examples are cytochrome c which forms the apoptosome together with APAF-1 and caspase-9, and Smac/Diablo which inhibits XIAP, which is an inhibitor of caspase-9.
Project:
Only a few functionally important proteins have been identified yet which translocate from the mitochondria to cytosol or vice versa during apoptosis. This project aims to find novel proteins and comprehends following methods established in our laboratory to detect them:
- Cell culture including SILAC
- Induction of apoptosis using Sorafenib
- Enrichment of mitochondrial and cytosolic fractions
- In-solution digestion of proteins with trypsin
- Analysis of tryptic peptides by nanoHPLC-mass spectrometry
- Protein identification and quantification using data analysis programs (MaxQuant)
- Validation of protein translocation by western blotting
More information about the proteomics group at IBV can be found at http://www.mn.uio.no/ibv/english/research/sections/bmb/groups/thiede/