Quantitative Phosphoproteomic Analysis of the Tumor Necrosis Factor Pathway

Protein phosphorylation has become a focus of many proteomic studies due to the central role that it plays in biology. We combine peptide-based gel-free isoelectric focusing and immobilized metal affinity chromatography to enhance the detection of phosphorylation events within complex protein samples using LC−MS. This method is then used to carry out a quantitative phosphoproteomic analysis of the tumor necrosis factor (TNF) pathway using HeLa cells metabolically labeled with <sup>15</sup>N-containing amino acids, where 145 phosphorylation sites were found to be up-regulated upon the activation of the TNF pathway. Keywords: isoelectric focusing • IMAC • phosphoproteome • metabolic labeling • TNF • PKA • apoptosis