Molden Rosalynn C, Goya Jonathan, Khan Zia, Garcia Benjamin A
Department of Chemistry, Frick Laboratory, Princeton University, Princeton, New Jersey 08544;
Mol Cell Proteomics. 2014 Apr;13(4):1106-18. doi: 10.1074/mcp.O113.036145. Epub 2014 Feb 16.
Signals that control responses to stimuli and cellular function are transmitted through the dynamic phosphorylation of thousands of proteins by protein kinases. Many techniques have been developed to study phosphorylation dynamics, including several mass spectrometry (MS)-based methods. Over the past few decades, substantial developments have been made in MS techniques for the large-scale identification of proteins and their post-translational modifications. Nevertheless, all of the current MS-based techniques for quantifying protein phosphorylation dynamics rely on the measurement of changes in peptide abundance levels, and many methods suffer from low confidence in phosphopeptide identification due to poor fragmentation. Here we have optimized an approach for the stable isotope labeling of amino acids by phosphate using [γ-¹⁸O₄]ATP in nucleo to determine global site-specific phosphorylation rates. The advantages of this metabolic labeling technique are increased confidence in phosphorylated peptide identification, direct labeling of phosphorylation sites, measurement phosphorylation rates, and the identification of actively phosphorylated sites in a cell-like environment. In this study we calculated approximate rate constants for over 1,000 phosphorylation sites based on labeling progress curves. We measured a wide range of phosphorylation rate constants from 0.34 min⁻¹ to 0.001 min⁻¹. Finally, we applied stable isotope labeling of amino acids by phosphate to identify sites that have different phosphorylation kinetics during G1/S and M phase. We found that most sites had very similar phosphorylation rates under both conditions; however, a small subset of sites on proteins involved in the mitotic spindle were more actively phosphorylated during M phase, whereas proteins involved in DNA replication and transcription were more actively phosphorylated during G1/S phase. The data have been deposited to the ProteomeXchange with the identifier PXD000680.
控制对刺激的反应和细胞功能的信号是通过蛋白激酶对数千种蛋白质的动态磷酸化来传递的。已经开发了许多技术来研究磷酸化动力学，包括几种基于质谱（MS）的方法。在过去几十年中，用于大规模鉴定蛋白质及其翻译后修饰的MS技术有了实质性的发展。然而，目前所有基于MS的定量蛋白质磷酸化动力学的技术都依赖于肽丰度水平变化的测量，并且由于碎片化不佳，许多方法在磷酸肽鉴定方面的可信度较低。在这里，我们优化了一种在细胞核中使用[γ-¹⁸O₄]ATP通过磷酸盐对氨基酸进行稳定同位素标记的方法，以确定全局位点特异性磷酸化速率。这种代谢标记技术的优点是提高了磷酸化肽鉴定的可信度、磷酸化位点的直接标记、磷酸化速率的测量以及在类似细胞环境中鉴定活跃磷酸化位点。在这项研究中，我们根据标记进展曲线计算了1000多个磷酸化位点的近似速率常数。我们测量了从0.34 min⁻¹到0.001 min⁻¹的广泛磷酸化速率常数。最后，我们应用磷酸盐对氨基酸的稳定同位素标记来鉴定在G1/S和M期具有不同磷酸化动力学的位点。我们发现大多数位点在两种条件下的磷酸化速率非常相似；然而，参与有丝分裂纺锤体的蛋白质上的一小部分位点在M期更活跃地磷酸化，而参与DNA复制和转录的蛋白质在G1/S期更活跃地磷酸化。数据已存入ProteomeXchange，标识符为PXD000680。
