Targeting hepatocytes from liver tissue by laser capture microdissection and proteomics expression profiling.
Förlag: Wiley Interscience
A tissue proteomics process is presented where hepatocyte cell isolation in combination with two-dimensional (2-D) gel electrophoresis and mass spectrometric identification were used to annotate the liver proteome. Laser microdissection of 8 m liver tissue sections was performed and protein expression profiling was compared using a variety of quantities of input cells, and gel separation conditions. The 30 m diameter laser generated the highest protein yields from the polymer coated caps following microsolubilization. We found that 6000 laser pulses (approximately 7200 hepatocytes) were required in order to generate high-resolution gel maps. Within homogeneous tissue samples, this could be accomplished in a total cycle time of 20 min using an automated dissection procedure. Close to 1000 high-quality gel annotations were generated from the corresponding 2-D gel expression profiles which matched closely the corresponding patterns of analytical-scale liver preparations detected by silver staining.
- Cell and Molecular Biology
- Matrix-assisted laser desorption/ionization-time of flight
- Laser microdissection
- Liver tissue
- Two-dimensional gel electrophoresis
- ISSN: 0173-0835