Relative quantification of erythropoietin receptor-dependent phosphoproteins using in-gel 18O-labeling and tandem mass spectrometry

On examining different proteomics approache for the investigation of structure‐function relationships of erythropoietin (EPO) receptor signaling, it was found that two‐dimensional gel electrophoresis/mass spectrometry procedures are clearly limited in their ability to detect low‐expressed signaling proteins. Instead it was found that a strategy involving anti‐phosphotyrosine immunoprecipitation, one‐dimensional gel electrophoresis (1DE), and capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) provides the sensitivity required for identification of signaling proteins. In the present work the immunoprecipitation/1DE/LC/MS approach was combined with an in‐gel 18O‐labeling technique to analyze EPO receptor‐dependent proteins. Identification and relative quantification of more than 180 EPO receptor‐dependent proteins were achieved directly based on the in‐gel 18O‐labeling approach. Copyright © 2005 John Wiley & Sons, Ltd.