OBJECTIVETo To establish a method of phosphoprotein affinity profiling for identifying phosphoproteomic differences between Thp-1 cells with or without lipopolysaccharide (LPS) stimulation, aiming to screen potential regulators involved in LPS pathway.

METHODSThp-1 cells were stimulated with 100 ng/ml PMA for 48 h to induce differentiation into mature macrophages, which, after culture for another 48 h in the absence of PMA, were either treated with 100 ng/ml LPS for 30 min or left untreated. After desalting procedure with ultrafiltration, the phosphoproteins enriched by phosphoprotein metal affinity column (PMAC) of both groups were run on 2-D electrophoresis to find the spots with different phosphorylation status. Finally, some of these spots were identified by mass spectrometry (MS) and subsequent bioinformatic analysis.

RESULTSCompared to untreated Thp-1 cells, LPS stimulated Thp-1 cells showed 29 spots with reproducible alterations on the 2-D map, including 8 representing up-regulated spots, 7 new spots, 10 down-regulated spots, and 4 absent spots. The newly emerged and absent protein spots were subjected to MS analysis, and 4 of them were identified to be involved in various cellular processes such as proteolysis, signal transduction and protein folding. Among these, phosphorylation of proteasome C2 subunit was dramatically up-regulated in LPS-stimulated cells, as was consistent with previous reports; the phosphorylation of Z-DNA-binding protein 1 has not been reported so far and needs further confirmation.

CONCLUSIONPhosphoprotein affinity profiling is an attractive method for screening novel regulators involved in LPS signaling pathways and can be widely used in systemic study of signal transduction.