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

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• 作者：Nieves Ibarrola ; Dario E. Kalume ; Mads Gronborg ; Akiko Iwahori ; and Akhilesh Pandey
• 刊名：Analytical Chemistry
• 出版年：2003
• 出版时间：November 15, 2003
• 年：2003
• 卷：75
• 期：22
• 页码：6043 - 6049
• 全文大小：172K
• 年卷期：v.75,no.22(November 15, 2003)
• ISSN：1520-6882

文摘

Posttranslational modifications are major mechanisms ofregulating protein activity and function in vertebrate cells.It is essential to obtain qualitative information aboutposttranslational modification patterns of proteins tounderstand signal transduction mechanisms in greaterdetail. However, it is equally important to measure thedynamics of posttranslational modifications such as phosphorylation to approach signaling networks from a systems biology perspective. Despite a number of advances,methods to quantitate posttranslational modificationsremain difficult to implement due to a number of factorsincluding lack of a generic method, elaborate chemicalsteps, and requirement for large amounts of sample. Wehave previously shown that stable isotope-containingamino acids in cell culture (SILAC) can be used todifferentially label growing cell populations for quantitation of protein levels. In this report, we extend the use ofSILAC as a novel proteomic approach for the relativequantitation of posttranslational modifications such asphosphorylation. We have used SILAC to quantitate theextent of known phosphorylation sites as well as to identifyand quantitate novel phosphorylation sites.