Objective: To develop a new method for analysis of protein phosphorylation modification in cultured neuron.
Methods: Cultured neurons were pre-incubated in DMEM without sodium phosphate for 15 min to deplete the metabolic pools. Neurons were then labeled with [32P] orthophosphate (2.78 x 10(6) Bq/ml) for 1.5 h and stimulated by either insulin (100 nmol/L), EGF (20 nm/L) or saline for 0, 5, 20, 60, 120 min. Reactions were terminated by freezing neurons in liquid nitrogen prior to the solubilizing of them in a lysis buffer containing 8 mol/L urea, 4% CHAPS, 2% Bio-lyte, pH 3-10, 2 mmol/L TBP. Protein concentrations were determined with Bio-Rad DC Protein Assay kit. The 32P-labled lysates isoelectrically focused on IPG Drystrip pH 3-10 or pH 4-7 Linear gels were subsequently separated in second-dimensional SDS-PAGE. The dried gel was autoradiographed for 5 days at -70 degrees C with an intensifying screen. Alternatively, the separated proteins were visualized by Coomassie Brilliant Blue (CBB) R250 straining.
Results: Autoradiography of the 2-DE-separated 32P-laebled neuron lysates revealed around 100 phosphoproteins. This phosphoprotein pattern was stable at 1.5 h after radiolabelling and did not vary significantly for up to 4 h further incubation in the absence of hormone. Most of the major proteins which are phosphorylated in response to insulin or EGF migrated with pH 4.6-6.5 and MW 20000-130000. Insulin and EGF induced similar but not identical patterns of protein phosphoryltion in neurons. Only a few phosphoproteins were abundant enough to be visualized by CBB straining, suggesting that abundance of these phosphoproteins is extremely low. Responses to both isulin and EGF are marked by more increased labeling of the constitutive phosphoproteins, compared with the appearance of new phosphoproteins.
Conclusion: This approach co-application of 32P-labeled with 2-DE separation and autoradiography has proven to be specific and sensitive in phosphoprotein analysis for neuron. It was valuable in functional proteomic analysis for protein phosphorylation modification during cellular signal transduction.