Yale Cancer Biology Institute, Yale University, West Haven, CT 06516, USA.
Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Dev Cell. 2021 Jan 11;56(1):111-124.e6. doi: 10.1016/j.devcel.2020.10.025. Epub 2020 Nov 24.
To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.
迄今为止，特定修饰类型和位置对蛋白质寿命的影响尚未得到系统说明。在这里，我们描述了一种蛋白质组学方法 DeltaSILAC，用于定量评估细胞内数千种蛋白质的特定位置磷酸化对其周转率的影响。基于准确且可重复的基于质谱的方法、细胞内使用稳定同位素标记的氨基酸的脉冲标记方法 (pSILAC)、磷酸化蛋白质组学和独特的肽级匹配策略，我们的 DeltaSILAC 分析揭示了许多磷酸化位点对蛋白质周转率的全局、意外的延迟效应。我们进一步发现，加速蛋白质周转率的磷酸化位点被选择性地用于细胞适应性，富含细胞周期蛋白依赖性激酶底物，并且在进化上保守，而围绕磷酸化位点的谷氨酸显著延迟蛋白质周转率。我们的方法代表了一种可推广的方法，并为在细胞信号转导和疾病生物学背景下确定磷酸化位点对蛋白质寿命的影响提供了丰富的资源。
