A differential phosphoproteomic analysis of retinoic acid-treated P19 cells.
External stimuli trigger internal signaling events within a cell that may represent either a temporary or permanent shift in the phosphorylation state of its proteome. Numerous reports have elucidated phosphorylation sites from a variety of biological samples and more recent studies have monitored the temporal dynamics of protein phosphorylation as a given system is perturbed. Understanding which proteins are phosphorylated as well as when they are phosphorylated may indicate novel functional roles within a system and allow new therapeutic avenues to be explored. To elucidate the dynamics of protein phosphorylation within differentiating murine P19 embryonal carcinoma cells, we induced P19 cells to differentiate using all-trans-retinoic acid and developed a strategy that combines isotopically labeled methyl esterification, immobilized metal affinity chromatography, mass spectrometric analysis, and a rigorous and unique data evaluation approach. We present the largest differential phosphoproteomic analysis using isotopically labeled methyl esterification to date, identifying a total of 472 phosphorylation sites on 151 proteins; 56 of these proteins had altered abundances following treatment with retinoic acid and approximately one-third of these have been previously associated with cellular differentiation. A series of bioinformatic tools were used to extract information from the data and explore the implications of our findings. This study represents the first global gel-free analysis that elucidates protein phosphorylation dynamics during cellular differentiation.
Smith
Jeffrey C
JC
Ottawa Institute of Systems Biology and Biochemistry, Microbiology and Immunology Department, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.
Duchesne
Marc A
MA
Tozzi
Pascal
P
Ethier
Martin
M
Figeys
Daniel
D
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D051379:Mice; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation / Q000187:drug effects; D025941:Protein Interaction Mapping; D020543:Proteome / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014212:Tretinoin / Q000494:pharmacology*
D010750:Phosphoproteins; D020543:Proteome; D014212:Tretinoin