Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals.
Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals.
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协调标签和分析策略，以获得完整成年动物的蛋白质周转率。
DOI：
10.1016/j.mcpro.2022.100252
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发表时间：
2022-07
影响因子：
7
通讯作者：
Lau, Edward
中科院分区：
文献类型：
--
作者：
Hammond, Dean E.;Simpson, Deborah M.;Franco, Catarina;Muelas, Marina Wright;Waters, John;Ludwig, R. W.;Prescott, Mark C.;Hurst, Jane L.;Beynon, Robert J.;Lau, Edward
蛋白质组中单个蛋白质丰度的变化可以通过调节蛋白质合成（新合成蛋白质输入蛋白质库的速率）或降解（蛋白质分子从库中去除的速率）引起。因此，对蛋白质组变化的充分理解需要定义这两个过程在蛋白质稳定中的作用，统称为蛋白质周转。由于蛋白质周转即使在库丰度没有明显变化的情况下也会发生，因此周转测量需要监测稳定同位素标记的前体通过蛋白质库的通量，例如标记的氨基酸或代谢前体，例如氯化铵或重水。在培养的细胞中，通过快速更换培养基来操纵前体池的能力很简单，但对于更复杂的系统，如完整的动物，这种方法变得更加复杂。个别方法会带来特定的并发症，不同方法的适用性尚未得到全面探讨。在这项研究中，我们比较了周转率的蛋白质在四个小鼠组织，从相同的近交系小鼠品系保持在相同的饲养条件下，测量使用[13 C6]赖氨酸或[2H2]O作为标记前体。我们表明，对于长寿命的蛋白质，这两种方法产生基本相同的措施的一阶速率常数降解。对于寿命短的蛋白质，需要通过前体池补偿赖氨酸的较慢平衡。我们评估不同的方法来提供这种补偿。我们的结论是，这两个标签是合适的，但仔细确定前体富集动力学的氨基酸标记是至关重要的，并有相当大的影响，数值上的衍生蛋白质周转率。蛋白质周转的重水或氨基酸标记的对照比较。氨基酸前体标记的延迟主要影响高周转蛋白质。两种方法在调整前体动力学后产生相似的周转率。动物蛋白质周转研究的分析工作流程建议。成年动物的蛋白质周转率与细胞培养有很大不同，需要仔细考虑前体平衡和可用性。这项研究由哈蒙德等人。比较了两种方法来测量蛋白质周转率的小鼠，在四个小鼠组织。用稳定同位素标记的氨基酸或重水标记有不同的分析复杂性，但优化后产生了可比的周转率。为动物蛋白质周转研究提供了分析和数据过滤建议。
Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [13C6]lysine or [2H2]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates. Controlled comparison of heavy water or amino acid labeling for protein turnover. Delays in amino acid precursor labeling mostly affect high turnover proteins Both methods produced similar turnover rates after adjustment of precursor kinetics. Recommendations for analytical workflows for protein turnover studies in animals. Protein turnover rates in adult animals differ greatly from cell culture and require careful considerations of precursor equilibration and availability. This study by Hammond et al. compared two approaches to measurement of protein turnover rates in the mouse, across four mouse tissues. Labeling with stable isotope–labeled amino acids or by heavy water had different analytical complications but yielded comparable turnover rates after optimization. Analytical and data filtering recommendations are provided for protein turnover studies in animals.
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影响因子：
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