A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture.
Posttranslational modifications are major mechanisms of regulating protein activity and function in vertebrate cells. It is essential to obtain qualitative information about posttranslational modification patterns of proteins to understand signal transduction mechanisms in greater detail. However, it is equally important to measure the dynamics of posttranslational modifications such as phosphorylation to approach signaling networks from a systems biology perspective. Despite a number of advances, methods to quantitate posttranslational modifications remain difficult to implement due to a number of factors including lack of a generic method, elaborate chemical steps, and requirement for large amounts of sample. We have previously shown that stable isotope-containing amino acids in cell culture (SILAC) can be used to differentially label growing cell populations for quantitation of protein levels. In this report, we extend the use of SILAC as a novel proteomic approach for the relative quantitation of posttranslational modifications such as phosphorylation. We have used SILAC to quantitate the extent of known phosphorylation sites as well as to identify and quantitate novel phosphorylation sites.
Ibarrola
Nieves
N
McKusick-Nathans Institute for Genetic Medicine and the Department of Biological Chemistry, Johns Hopkins University, Baltimore, Maryland 21287, USA.
Kalume
Dario E
DE
Gronborg
Mads
M
Iwahori
Akiko
A
Pandey
Akhilesh
A
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D006801:Humans; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013997:Time Factors
D002247:Carbon Isotopes; D011506:Proteins; D008239:Lysine