Concomitant determination of absolute values of cellular protein amounts, synthesis rates, and turnover rates by quantitative proteome profiling
Concomitant determination of absolute values of cellular protein amounts, synthesis rates, and turnover rates by quantitative proteome profiling
复制标题
DOI：
10.1074/mcp.m200026-mcp200
复制
发表时间：
2002-07-01
影响因子：
7
通讯作者：
Mikulits, W
中科院分区：
文献类型：
--
作者：
Gerner, C;Vejda, S;Mikulits, W
从S-35放射性标记细胞中分离的蛋白质组分的二维凝胶电泳提供了细胞内数量，S-35掺入率，蛋白质修饰和多达数千个单个蛋白质的亚细胞定位的定性信息。在这项研究中，我们扩展了蛋白质组分析，以提供单个蛋白质合成速率的定量数据。我们将放射标记蛋白的荧光检测与SYPRO ruby（TM）染色和随后的相同凝胶的放射自显影相结合，从而定量蛋白质量和35S掺入。为了校正绝对合成速率的计算，我们测定了经白细胞介素-6预处理的HepG2细胞分泌的放射性标记的触珠蛋白的量和放射自显影强度。这使我们能够获得S-35并入每个自射线照相强度单位的标准校准值。该值用于热休克U937细胞时间过程实验中蛋白质合成速率的测定。我们测量了hsp70的增加量，并通过对确定的hsp70合成速率随时间的积分来计算。两种方法得到的结果相似，验证了我们的标准化程序。基于蛋白质在细胞代谢稳定状态下的合成速率基本上可以补偿蛋白质的降解这一假设，我们通过二维凝胶测定的蛋白质量和合成速率计算了蛋白质的生物半衰期。计算出的蛋白质半衰期与脉冲追逐实验的半衰期接近，从而验证了这种新方法。总之，我们设计了一种方法来评估定量蛋白质组谱，包括确定单个数量，合成和蛋白质周转率。
Two-dimensional gel electrophoresis of protein fractions isolated from S-35-radiolabeled cells provides qualitative information on intracellular amounts, S-35 incorporation rates, protein modifications, and subcellular localizations of up to thousands of individual proteins. In this study we extended proteome profiling to provide quantitative data on synthesis rates of individual proteins. We combined fluorescence detection of radiolabeled proteins with SYPRO ruby(TM) staining and subsequent autoradiography of the same gels, thereby quantifying protein amounts and 35S incorporation. To calibrate calculation of absolute synthesis rates, we determined the amount and autoradiograph intensity of radiolabeled haptoglobin secreted by interleukin-6 pretreated HepG2 cells. This allowed us to obtain a standard calibration value for S-35 incorporation per autoradiograph intensity unit. This value was used to measure protein synthesis rates during time course experiments of heat-shocked U937 cells. We measured the increasing amounts of hsp70 and calculated it by integration of the determined hsp70 synthesis rates over time. Similar results were obtained by both methods, validating our standardization procedure. Based on the assumption that the synthesis rate of proteins in a steady state of cell metabolism would essentially compensate protein degradation, we calculated biological half-lives of proteins from protein amounts and synthesis rates determined from two-dimensional gels. Calculated protein half-lives were found close to those determined by pulse-chase experiments, thus validating this new method. In conclusion, we devised a method to assess quantitative proteome profiles covering determination of individual amounts, synthesis, and turnover rates of proteins.