Qualitative and Quantitative Analysis of Phosphoproteomic Experimental Workflow Based on Phosphoprotein Enrichment Strategy and Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) Techniques

The purpose of this research work was to investigate whether such a workflow including the phosphoproteins enriched by affinity purification, isolated and quantified by two-dimensional difference gel electrophoresis, the differential protein spots identified by mass spectrometry, was suitable to dissect the intact phosphoprotein profile that mediated cell signaling pathways. Endothelial cells with or without vascular endothelial growth factor (VEGF) induction were used in the experiment. The accidental error introduced by enrichment column was assayed by control-to-control 2D-DIGE analysis and the efficiency of the workflow was tested by control-to-sample 2D-DIGE analysis. The data indicated that a high level of accidental error was introduced by different phosphoprotein enrichment columns and could be reduced by slowing down the flow rate of the mobile phase at the expense of sensitivity and specificity of enrichment column. Dephosphorylation did not occur for most high abundance phosphoproteins based on the sensitivity of 2D-DIGE detection. The advantage of this workflow was that multiple phosphorylated proteins could be visualized on the 2D-gel directly, but DIGE minimal label only quantified the limited high or medium abundance phosphoproteins. The sensitivity and accuracy of 2D-DIGE measurement were still not good enough to dissect the intact phosphoprotein profile that involved in cell signal pathway. Consequently, phosphoproteomic laboratory workflow based on phosphoproteins enrichment strategy combined with 2D-DIGE quantification does not have enough sensitivity and accuracy to dissect the intact phosphoprotein profile that mediated cell signaling pathways. Low abundance and ionization of the phosphopeptides are the main reasons that lead to the failure of protein identification even if the phosphoproteins were enriched.