Quantitative analysis of brain nuclear phosphoproteins identifies developmentally regulated phosphorylation events.
Protein phosphorylation is a globally adopted and tightly controlled post-translational modification, and represents one of the most important molecular switching mechanisms that govern the entire spectrum of biological processes. In the central nervous system, it has been demonstrated that phosphorylation of key proteins mediating chromatin remodeling and gene transcription plays an important role controlling brain development, synaptogenesis, learning and memory. Many studies have focused on large scale identification of phosphopeptides in brain tissue. These studies have identified phosphorylation site specific motifs useful for predicting protein kinase substrates. In this study, we applied a previously developed quantitative approach, stable isotope labeling of amino acids in mammals (SILAM), to quantify changes in the phosphorylation of nuclear proteins between a postnatal day one (p1) and a p45 rat brain cortex. Using a 15N labeled rat brain as an internal standard, we quantified 705 phosphopeptides in the p1 cortex and 1477 phosphopeptides in the p45 cortex, which translates to 380 and 585 phosphoproteins in p1 and p45 cortex, respectively. Bioinformatic analysis of the differentially modified phosphoproteins revealed that phosphorylation is upregulated on multiple components of chromatin remodeling complexes in the p1 cortex. Taken together, we demonstrated for the first time the usefulness of employing stable isotope labeled rat tissue for global quantitative phosphorylation analysis.
Liao
Lujian
L
Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA.
McClatchy
Daniel B
DB
Park
Sung Kyu
SK
Xu
Tao
T
Lu
Bingwen
B
Yates
John R
JR
D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000254:growth & development* / Q000378:metabolism*; D001923:Brain Chemistry; D002525:Cerebellar Cortex / Q000737:chemistry; D019295:Computational Biology / Q000379:methods; D007553:Isotope Labeling; D009687:Nuclear Proteins / Q000032:analysis*; D010748:Phosphopeptides / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D015203:Reproducibility of Results
D009687:Nuclear Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins