10.1021/pr050270m.s006 Greg T. Cantin Greg T. Cantin John D. Venable John D. Venable Daniel Cociorva Daniel Cociorva John R. Yates John R. Yates Quantitative Phosphoproteomic Analysis of the Tumor Necrosis Factor Pathway American Chemical Society 2006 isoelectric 145 phosphorylation sites HeLa cells metabolically TNF pathway LC Tumor Necrosis Factor Pathway Protein phosphorylation PKA proteomic studies metal affinity chromatography phosphorylation events tumor necrosis factor phosphoproteomic analysis Quantitative Phosphoproteomic Analysis IMAC protein samples 2006-01-06 00:00:00 Dataset https://acs.figshare.com/articles/dataset/Quantitative_Phosphoproteomic_Analysis_of_the_Tumor_Necrosis_Factor_Pathway/3245521 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