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，尚未系统地说明特定修饰类型和位点对蛋白质寿命的影响。在这里，我们描述了一种蛋白质组学方法 DeltaSILAC，以定量评估位点特异性磷酸化对活细胞中数千种蛋白质周转的影响。基于准确且可重现的基于质谱的方法、使用细胞中稳定同位素标记氨基酸 (pSILAC) 的脉冲标记方法、磷酸蛋白质组学和独特的肽水平匹配策略，我们的 DeltaSILAC 分析揭示了一种全局的、意想不到的延迟效应许多磷酸盐对蛋白质周转的影响。我们进一步发现加速蛋白质周转的磷酸化位点在功能上被选择用于细胞适应性，富含细胞周期蛋白依赖性激酶底物，并且在进化上是保守的，而磷酸位点周围的谷氨酸会显着延迟蛋白质周转。我们的方法代表了一种可推广的方法，并为在细胞信号传导和疾病生物学的背景下优先考虑磷酸化位点对蛋白质寿命的影响提供了丰富的资源。