为可靠的蛋白质组分析确定细胞培养条件的特征:A549、分化的 THP-1 和 NR8383 细胞系的体外研究

IF 4.8 2区 医学 Q1 TOXICOLOGY
Rico Ledwith, Tobias Stobernack, Antje Bergert, Aileen Bahl, Mario Pink, Andrea Haase, Verónica I. Dumit
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引用次数: 0

摘要

蛋白质组学研究产生了高维数据集,但由于实验设置不同,差异很大,因此无法对不同研究进行整合或比较。此外,细胞培养条件也会对研究结果产生巨大影响。本研究系统地探讨了实验参数对常用细胞系--A549、分化的 THP-1 巨噬细胞样细胞和 NR8383--毒性研究蛋白质组谱的影响。这项工作的重点是分析不同容器、细胞通过数和分化后收获时间等细胞培养设置在蛋白质组水平上的影响,旨在提高用于危害评估的蛋白质组分析的可靠性。我们利用基于质谱仪的蛋白质组学,通过无标记方法对蛋白质进行了精确定量。我们的研究结果表明,在不同设置下培养细胞时,蛋白质组会发生显著变化。对这些变化的进一步分析表明,它们与蛋白质错误折叠、氧化应激和蛋白酶体活性相关的特定细胞通路有关。相反,细胞通过数对蛋白质组的影响很小,这表明进行可重复生物复制的范围是可靠的。值得注意的是,随着时间的推移,dTHP-1 细胞分化后的蛋白质组发生了重大改变,尤其是对巨噬细胞功能至关重要的通路产生了影响。这一发现是解释实验结果的关键。这些结果突出表明,在基于蛋白质组学的治疗效果评估中,需要标准化的培养条件,以确保结果的可靠性,这是蛋白质组学数据获得监管部门认可的前提条件。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Towards characterization of cell culture conditions for reliable proteomic analysis: in vitro studies on A549, differentiated THP-1, and NR8383 cell lines

Towards characterization of cell culture conditions for reliable proteomic analysis: in vitro studies on A549, differentiated THP-1, and NR8383 cell lines

Proteomic investigations result in high dimensional datasets, but integration or comparison of different studies is hampered by high variances due to different experimental setups. In addition, cell culture conditions can have a huge impact on the outcome. This study systematically investigates the impact of experimental parameters on the proteomic profiles of commonly used cell lines—A549, differentiated THP-1 macrophage-like cells, and NR8383—for toxicity studies. The work focuses on analyzing the influence at the proteome level of cell culture setup involving different vessels, cell passage numbers, and post-differentiation harvesting time, aiming to improve the reliability of proteomic analyses for hazard assessment. Mass-spectrometry-based proteomics was utilized for accurate protein quantification by means of a label-free approach. Our results showed that significant proteome variations occur when cells are cultivated under different setups. Further analysis of these variations revealed their association to specific cellular pathways related to protein misfolding, oxidative stress, and proteasome activity. Conversely, the influence of cell passage numbers on the proteome is minor, suggesting a reliable range for conducting reproducible biological replicates. Notable, substantial proteome alterations occur over-time post-differentiation of dTHP-1 cells, particularly impacting pathways crucial for macrophage function. This finding is key for the interpretation of experimental results. These results highlight the need for standardized culture conditions in proteomic-based evaluations of treatment effects to ensure reliable results, a prerequisite for achieving regulatory acceptance of proteomics data.

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来源期刊
Archives of Toxicology
Archives of Toxicology 医学-毒理学
CiteScore
11.60
自引率
4.90%
发文量
218
审稿时长
1.5 months
期刊介绍: Archives of Toxicology provides up-to-date information on the latest advances in toxicology. The journal places particular emphasis on studies relating to defined effects of chemicals and mechanisms of toxicity, including toxic activities at the molecular level, in humans and experimental animals. Coverage includes new insights into analysis and toxicokinetics and into forensic toxicology. Review articles of general interest to toxicologists are an additional important feature of the journal.
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