A Proteomic Approach for Quantitation of Phosphorylation Using Stable Isotope Labeling in Cell Culture

• Published in: Analytical chemistry (Washington) Vol. 75; no. 22; pp. 6043 - 6049
• Main Authors: Ibarrola, Nieves, Kalume, Dario E, Gronborg, Mads, Iwahori, Akiko, Pandey, Akhilesh
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.11.2003
• Subjects: Amino Acid Sequence; Amino acids; Biological and medical sciences; Carbon Isotopes; Cell culture; Cell Culture Techniques - methods; Cell Line; Cells; Diverse techniques; Fundamental and applied biological sciences. Psychology; Humans; Isotopes; Lysine - chemistry; Mass Spectrometry - methods; Molecular and cellular biology; Molecular Sequence Data; Phosphorus; Phosphorylation; Protein Processing, Post-Translational; Proteins - chemistry; Proteins - metabolism; proteomics; Proteomics - methods; Q1; Quantitation; Signal transduction; Time Factors; Posttranslational modification; Cell culture; Phosphorylation; Isotope labelling; Aminoacid; Proteomics; Mass spectrometry; Protein; Quantitative analysis
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac034931f

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.