Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington Avenue SE, Minneapolis, Minnesota 55455, USA.
J Proteome Res. 2010 May 7;9(5):2087-97. doi: 10.1021/pr9007738.
Current techniques for quantitative proteomics focus mainly on measuring overall protein dynamics, which is the net result of protein synthesis and degradation. Understanding the rate of this synthesis/degradation is essential to fully appreciate cellular dynamics and bridge the gap between transcriptome and proteome data. Protein turnover rates can be estimated through "label-chase" experiments employing stable isotope-labeled precursors; however, the implicit assumption of steady-state in such analyses may not be applicable for many intrinsically dynamic systems. In this study, we present a novel extension of the "label-chase" concept using SILAC and a secondary labeling step with iTRAQ reagents to estimate protein turnover rates in Streptomyces coelicolor cultures undergoing transition from exponential growth to stationary phase. Such processes are of significance in Streptomyces biology as they pertain to the onset of synthesis of numerous therapeutically important secondary metabolites. The dual labeling strategy enabled decoupling of labeled peptide identification and quantification of degradation dynamics at MS and MS/MS scans respectively. Tandem mass spectrometry analysis of these multitagged proteins enabled estimation of degradation rates for 115 highly abundant proteins in S. coelicolor. We compared the rate constants obtained using this dual labeling approach with those from a SILAC-only analysis (assuming steady-state) and show that significant differences are generally observed only among proteins displaying considerable temporal dynamics and that the directions of these differences are largely consistent with theoretical predictions.
目前的定量蛋白质组学技术主要集中在测量整体蛋白质动力学上，这是蛋白质合成和降解的净结果。了解这种合成/降解的速度对于充分了解细胞动力学以及弥合转录组和蛋白质组数据之间的差距至关重要。可以通过使用稳定同位素标记前体的“标记追踪”实验来估计蛋白质周转率；然而，这种分析中隐含的稳态假设可能不适用于许多内在动态系统。在这项研究中，我们使用 SILAC 和 iTRAQ 试剂的二次标记步骤对“标记追踪”概念进行了新颖的扩展，以估计从指数生长到静止期过渡的变铅青链霉菌培养物中的蛋白质周转率。这些过程在链霉菌生物学中具有重要意义，因为它们涉及到许多治疗上重要的次生代谢物合成的开始。双重标记策略分别能够在 MS 和 MS/MS 扫描中解耦标记肽的鉴定和降解动力学的定量。对这些多标记蛋白的串联质谱分析使我们能够估计变铅青链霉菌中 115 种高丰度蛋白的降解速率。我们将使用这种双重标记方法获得的速率常数与仅使用 SILAC（假设稳态）获得的速率常数进行了比较，并表明通常仅在显示出相当大的时间动力学的蛋白质之间观察到显著差异，并且这些差异的方向与理论预测基本一致。
