A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture
A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture
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DOI：
10.1021/ac034931f
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发表时间：
2003-11-15
影响因子：
7.4
通讯作者：
Pandey, A
中科院分区：
文献类型：
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作者：
Ibarrola, N;Kalume, DE;Pandey, A
翻译后修饰是调节脊椎动物细胞中蛋白质活性和功能的主要机制。获得有关蛋白质翻译后修饰模式的定性信息对于更详细地了解信号转导机制至关重要。然而，测量磷酸化等翻译后修饰的动态，从系统生物学的角度探讨信号网络也同样重要。尽管取得了许多进展，但由于缺乏通用方法、复杂的化学步骤以及需要大量样品等多种因素，定量翻译后修饰的方法仍然难以实施。我们之前已经证明，细胞培养物中含有稳定同位素的氨基酸 (SILAC) 可用于差异标记生长的细胞群，以定量蛋白质水平。在本报告中，我们扩展了 SILAC 作为一种新型蛋白质组学方法的用途，用于磷酸化等翻译后修饰的相对定量。我们使用 SIIAC 来定量已知磷酸化位点的范围以及识别和定量新的磷酸化位点。
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 SIIAC to quantitate the extent of known phosphorylation sites as well as to identify and quantitate novel phosphorylation sites.