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 15N-containing amino acids, where 145 phosphorylation sites were found to be up-regulated upon the activation of the TNF pathway.
Cantin
Greg T
GT
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd. SR11, La Jolla, California 92037, USA.
Venable
John D
JD
Cociorva
Daniel
D
Yates
John R
JR
D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D006367:HeLa Cells; D006801:Humans; D007525:Isoelectric Focusing; D007553:Isotope Labeling; D013058:Mass Spectrometry; D009587:Nitrogen Isotopes / Q000032:analysis / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010748:Phosphopeptides / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome; D014409:Tumor Necrosis Factor-alpha