Pacific Northwest National Laboratory , Richland, Washington 99354, United States.
Anal Chem. 2017 Dec 19;89(24):13559-13566. doi: 10.1021/acs.analchem.7b03984. Epub 2017 Dec 6.
Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n = 5) were fed C-labeled lysine ("heavy") feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed ("light"), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins and quantify heavy/light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ∼70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing turnover measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein biomarkers through better understanding of processes governing biomarker kinetics.
蛋白质周转对于细胞和生物体的整体健康非常重要，它为替换旧的、受损的或功能失调的蛋白质提供了一种策略。蛋白质周转还可以提供生物标志物动力学的信息，因为更好地了解蛋白质的合成和降解可以提高生物标志物的临床应用价值。在这里，使用脉冲追踪稳定同位素标记实验在体内测量了大鼠血浆蛋白的周转率。在脉冲期间，大鼠（n = 5）用 C 标记的赖氨酸（“重”）饲料喂养 23 天，以标记蛋白质。在追踪期间，饲料被换成等量的未标记饲料（“轻”），并在 28 天内从大鼠身上重复采集 10 个时间点的血液样本。血浆样品用胰蛋白酶消化，并通过液相色谱-串联质谱（LC-MS/MS）进行分析。MaxQuant 用于鉴定肽和蛋白质，并定量重/轻赖氨酸比。使用常微分方程系统计算蛋白质周转率。使用这种方法，鉴定了 273 种蛋白质，并对 157 种血浆蛋白质的周转率进行了定量，半衰期范围为 0.3-103 天。对于约 70 种最丰富的蛋白质，大鼠之间的周转率变异性较低（中位数变异系数：0.09）。应用基于活性的蛋白质分析方法，使用氟膦酸盐（FP2）活性探针对混合血浆样品进行富集丝氨酸水解酶。这种富集导致另外 17 种蛋白质的周转率增加。这项研究首次在体内测量了大鼠的整体血浆蛋白质周转率，测量了任何动物模型中蛋白质周转率的变异性，并利用基于活性的蛋白质分析方法增强了靶向、低丰度蛋白质（如常用作生物标志物的蛋白质）的周转率测量。测量的蛋白质周转率对于理解蛋白质周转率在细胞和生物体健康中的作用以及通过更好地理解控制生物标志物动力学的过程提高蛋白质生物标志物的应用价值非常重要。
