title: Initial analysis of the phosphoproteome of Chinese hamster ovary cells using electrophoresis.
abstract: Protein phosphorylation is a common post-translational modification of enormous biological importance. Analysis of phosphorylation at the global level should shed light on the use of this modification to regulate metabolism, signal transduction, and other processes. We have begun a proteomic analysis of phosphorylation using two-dimensional gel electrophoresis. Chinese hamster ovary (CHO) cells were metabolically labeled using 32P-orthophosphate. The proteins were extracted and run on two-dimensional electrophoresis. Gels were stained using colloidal Coomassie stain, dried, and phosphorimaged. The Coomassie stain allowed the observation of 468 individual protein spots. The phosphorimage showed 181 spots. The phosphoproteome of CHO cells therefore comprises around one third as many proteins as the CHO cell abundance proteome. However, the most intense spots in the phosphoproteome usually do not correlate with intense spots in the abundance proteome. We investigated the effects of labeling time, finding that the number of observable spots increases but the relative intensities also change. We also investigated the effects of adding a phosphatase inhibitor during labeling. Finally, we evaluated a phosphoprotein-specific stain (Pro-Q Diamond) in comparison with radiolabeling methods. There is not perfect correlation between radiolabeled phosphoproteins and Pro-Q Diamond-stained phosphoproteins.
authors.affiliation: Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA.
authors.forename: ZhaoYuan
authors.initials: Z
authors.lastname: Chen
authors.forename: Katie
authors.initials: K
authors.lastname: Southwick
authors.forename: Craig D
authors.initials: CD
authors.lastname: Thulin
mesh_terms: D000818:Animals; D016466:CHO Cells; D002623:Chemistry Techniques, Analytical; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional; D010750:Phosphoproteins / Q000737:chemistry*; D010761:Phosphorus Radioisotopes; D020543:Proteome / Q000737:chemistry*
chemical_list: D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D020543:Proteome