A quantitative spatial proteomics analysis of proteome turnover in human cells.
A quantitative spatial proteomics analysis of proteome turnover in human cells.
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DOI：
10.1074/mcp.m111.011429
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发表时间：
2012-03
期刊：
影响因子：
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通讯作者：
Lamond AI
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作者：
Boisvert FM;Ahmad Y;Gierliński M;Charrière F;Lamont D;Scott M;Barton G;Lamond AI
在后基因组时代，在整个蛋白质组水平上测量内源性细胞蛋白质的性质，如表达水平、亚细胞定位和周转率仍然是一个重大挑战。用于测量mRNA表达的定量方法不能可靠地预测相应的蛋白质水平，并且提供很少或没有关于其他蛋白质性质的信息。在这里，我们描述了一个组合脉冲标记，空间蛋白质组学和数据分析策略来表征内源性表达，未标记的人类蛋白质在不同的亚细胞区室的表达，定位，合成，降解和周转率。使用定量质谱和稳定同位素标记的氨基酸在细胞培养物中，共80,098肽从8,041 HeLa蛋白质进行了定量，并确定其在细胞质，细胞核和核仁之间的空间分布和可视化使用专门的软件工具开发的PepTracker。使用信息从离子强度和同位素比的变化率，蛋白质丰度水平和蛋白质合成，降解和周转率计算为整个细胞和各自的细胞质，细胞核，和核仁室。内源性HeLa蛋白的表达水平变化高达7个数量级。HeLa蛋白的平均周转率为1.20 h。周转率与分子量或净电荷均不相关，但与丰度相关，高丰度蛋白质的半衰期长于平均值。快周转蛋白的PEST基序的频率总体上高于慢周转蛋白，但没有观察到氨基或羧基末端氨基酸的身份和周转率之间的一般相关性。一个子集的蛋白质被确定存在于不同的周转率取决于其亚细胞定位池。这与大的多蛋白复合物的亚基密切相关，表明它们的组装是在与其主要功能部位不同的亚细胞位置控制的一般机制。
Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ∼20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.