Two-dimensional electrophoresis reveals differential protein expression in high- and low-secreting variants of the rat basophilic leukaemia cell line.
The aim of this investigation was the identification of cellular proteins that confer a high secretory phenotype on subclones of the rat basophilic leukaemia (RBL) cell line as a model of mast cell regulated degranulation. Following protein separation by two-dimensional (2-D) electrophoresis and silver staining, more than 2000 polypeptide "spots" were resolved reproducibly. Higher sample loads and Coomassie blue staining facilitated the identification by delayed extraction-matrix-assisted laser desorption/ionization (DE-MALDI) mass spectrometry of several polypeptides that were differentially expressed in the high- and low-secreting clones. Several proteins were identified whose expression could contribute to the difference in secretory phenotype. Furthermore, silver-stained 2-D gel patterns suggested differential expression of proteins in the 20-25 kDa and the pI 4.5-7.5 range, characteristic of small guanosine 5'-triphosphate (GTP)-binding proteins. By a combination of "GTP overlay" and immunoblotting, we were able to demonstrate differential expression of small GTP binding-proteins, including Rab3 proteins, in high-and low-secreting clones. The sensitivity of this complementary approach facilitated the detection of some GTP binding and Rab3 proteins, whose expression was not evident in silver-stained 2-D gels.
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, UK. k.carroll@sheffield.ac.uk
K
K
Carroll
K
K
Ray
B
B
Helm
E
E
Carey
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D019204:GTP-Binding Proteins / Q000032:analysis*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007120:Immunochemistry; D007553:Isotope Labeling; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000032:analysis*; D010761:Phosphorus Radioisotopes; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014407:Tumor Cells, Cultured; D020692:rab3 GTP-Binding Proteins / Q000032:analysis
D009363:Neoplasm Proteins; D010761:Phosphorus Radioisotopes; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins