Diabetes Genome Anatomy Project |
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Projects > Project 4
Project 4:
Information of levels, modification states and
subcellular distribution of proteins in cells and tissues
Primary Investigator: Silvia Corvera, M.D. (U. Mass Medical Center)
Co-Investigator: John Leszyk, Ph.D. (U. Mass Medical Center)
[ Proteomic Mass Spectrometry Lab at U. Mass Medical Center ]
Summary
The role of project 4 and the proteomics core facility is to complement the DGAP with information regarding the levels, modification states and subcellular distribution of proteins in cells and tissues that are being analyzed by gene chip methodologies.
The discovery aspect of this project focuses on the identification of novel targets or molecular processes that are rapidly induced by insulin in adipose cells. The underlying hypothesis for this project is that insulin action will cause rapid changes in the subcellular localization and/or oligomerization state of key regulatory proteins and protein complexes, and that such changes can be detected using a comprehensive approach that combines subcellular fractionation, detergent solubilization, velocity gradient centrifugation and Mass spectroscopy. The hypothesis is based on key discoveries in signal transduction, which demonstrate the rapid formation of complexes stabilized by interactions such as that of SH2 domains with phosophotyrosine or PH domains with phosphoinositides rapidly in response to receptor activation. Furthermore, the highly increased sensitivity of protein detection methodology combined with mass spectrometry and database correlation analysis allows a comprehenesive analysis and documentation of such changes.
Specific Aims
The first phase of project 4 aims at detecting and identifying major oligomeric complexes and cytosol-membrane translocations formed in response to acute insulin action in 3T3-L1 adipocytes. Detailed methods for each step are described in the Methods and Protocols Section. Briefly, the protocol consists in:
- Differentiation of post confluent 3T3-L1 fibroblasts using dexamethasone, methyl-isobutyl-xanthine and insulin.
- Fully differentiated cells areincubated in the absence or presence of insulin (100nM) for 5 minutes at 37oC,
- Cells are homogenized and subcellular fractions (cytosol, peripheral membrane/cytoskeletal proteins, membrane/cytoskeletal proteins) are obtained.
- Each subcellular fraction is subjected to velocity sedimentation. Fractions are collected and run side-by-side on SDS-polyacrulamide gels.
- Gels are stained with Sypro-Ruby and photographed.
- Bands displaying different intensities between control and insulin-treated conditions at any point in the gradient are excised.
- Excised bands are subjected to mass spectrometry, and proteins within the bands identified by database correlation analysis.
Two examples of results being generated by this protocol are illustrated:
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| Example 1 | Example 2 |
References
- C. Borchers, Peter J. F., Hall M. C., Kunkel T. A.,Tomer K. B., Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer. Anal Chem, 72 p. 1163-8. (2000).
- K. Gevaert,Vandekerckhove J., Protein identification methods in proteomics. Electrophoresis, 21 p. 1145-54. (2000).
- A. Shevchenko, Loboda A., Ens W.,Standing K. G., MALDI quadrupole time-of-flight mass spectrometry: a powerful tool for proteomic research. Anal Chem, 72 p. 2132-41. (2000).
Protocols
- Isolation and Analysis of sub-cellular Fractions from 3T3L1 Adipocytes
- Isolation and Analysis of Sub-cellular Fractions from Small and Large Adipocytes from Wildtype and Firko Mice