Stobernack Tobias, Dommershausen Nils, Alcolea-Rodríguez Víctor, Ledwith Rico, Bañares Miguel A, Haase Andrea, Pink Mario, Dumit Verónica I
German Federal Institute for Risk Assessment (BfR), Department of Chemical and Product Safety, Max-Dohrn-Straße 8-10, 10589, Berlin, Germany.
Spanish National Research Council - Institute of Catalysis and Petrochemistry (ICP-CSIC), Spectroscopy and Industrial Catalysis group, Marie Curie, 2, Madrid, 28049, Spain.
Small Methods. 2024 Dec;8(12):e2400420. doi: 10.1002/smtd.202400420. Epub 2024 May 30.
Proteomic investigations yield high-dimensional datasets, yet their application to large-scale toxicological assessments is hindered by reproducibility challenges due to fluctuating measurement conditions. To address these limitations, this study introduces an advanced tandem mass tag (TMT) labeling protocol. Although labeling approaches shorten data acquisition time by multiplexing samples compared to traditional label-free quantification (LFQ) methods in general, the associated costs may surge significantly with large sample sets, for example, in toxicological screenings. However, the introduced advanced protocol offers an efficient, cost-effective alternative, reducing TMT reagent usage (by a factor of ten) and requiring minimal biological material (1 µg), while demonstrating increased reproducibility compared to LFQ. To demonstrate its effectiveness, the advanced protocol is employed to assess the toxicity of nine benchmark nanomaterials (NMs) on A549 lung epithelial cells. While LFQ measurements identify 3300 proteins, they proved inadequate to reveal NM toxicity. Conversely, despite detecting 2600 proteins, the TMT protocol demonstrates superior sensitivity by uncovering alterations induced by NM treatment. In contrast to previous studies, the introduced advanced protocol allows simultaneous and straightforward assessment of multiple test substances, enabling prioritization, ranking, and grouping for hazard evaluation. Additionally, it fosters the development of New Approach Methodologies (NAMs), contributing to innovative methodologies in toxicological research.
蛋白质组学研究产生高维数据集，然而由于测量条件波动导致的可重复性挑战阻碍了其在大规模毒理学评估中的应用。为解决这些局限性，本研究引入了一种先进的串联质谱标签（TMT）标记方案。虽然与传统的无标记定量（LFQ）方法相比，标记方法通过对样品进行多路复用缩短了数据采集时间，但对于大型样本集（例如在毒理学筛查中），相关成本可能会大幅飙升。然而，引入的先进方案提供了一种高效、经济高效的替代方案，减少了TMT试剂的使用量（减少了十分之九），所需生物材料最少（1微克），同时与LFQ相比具有更高的可重复性。为证明其有效性，采用先进方案评估了九种基准纳米材料（NMs）对A549肺上皮细胞的毒性。虽然LFQ测量鉴定出3300种蛋白质，但事实证明它们不足以揭示纳米材料的毒性。相反，尽管TMT方案检测到2600种蛋白质，但通过发现纳米材料处理引起的变化，它显示出更高的灵敏度。与先前的研究不同，引入的先进方案允许同时直接评估多种测试物质，从而能够对危害评估进行优先级排序、排名和分组。此外，它促进了新方法学（NAMs）的发展，为毒理学研究中的创新方法做出了贡献。
