A combined SIMAC-HILIC-TiO_(2) strategy for large-scale phosphoproteomic analysis of sub-milligram amounts of sample material

摘要

The combination of sub-cellular fractionation, SIMAC, HILIC, and TiO_(2) enrichment resulted in the identification of more than 4600 unique phosphopeptides from < 500(mu)g of protein from a SILAC labeled HeLa cell lysate after 5 min. of EGF stimulation (1.0percent FDR). Phosphoproteomic analysis perfomed on starved or non-starved cells showed a similar number of phosphopeptide identifications. The phosphorylation increase in response to EGF, however, seemed to be higher in the starved cells, even though many known effector of EGF signaling could be identified to increase in phosphorylation level in the non-starved cells.