Diabetes Genome Anatomy Project |
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Projects > Project 8
Project 8:
Signal-dependent Interactions in Insulin Action
Primary Investigator: Matthias Mann, Ph.D. (University of Southern Denmark)
Specific Aims
- Measure proteome-wide changes in phosphorylation triggered by insulin in white and brown adipocyte cell lines.
- Compare changes of the insulin-induced tyrosine phosphoproteome in normal brown adipocytes and in cells with specific knock out for key insulin signaling factors.
- Identify proteins interacting specifically with insulin-induced phosphotyrosine moieties.
Summary
Insulin binding to its receptor initiates a complex network of events, starting with a tyrosine phosphorylation cascade that branches out to affect multiple endpoints. It is likely that many steps of the insulin pathway are adversely affected in various forms of type II diabetes. However, the polygenic nature of the disease makes it difficult to understand its molecular level. While gene expression methods are very well developed, they do not address many of the changes that may occur in cell signaling. To study changes in protein abundance, we are using a combination of nano-flow high performance liquid chromatography, sensitive mass spectrometers (LTQ-FTMS), and the SILAC method (Stable Isotope Labeling by Amino acids in Cell culture). These techniques will allow us to define changes in the insulin-dependent tyrosine phosphorylation on a proteomic scale.
Summary and Protocols Document
The above document contains the project specific aims, summary and the following protocols:- Culture of brown adipocytes (BAT) in SILAC Medium and Insulin induction
- Immunoprecipitation
- In Gel digestion
- Sample preparation after "in gel" digestion
- HPLC system and mass spectrometer