Oxidative stress, a commonly used paradigm to explain nanoparticle (NP)-induced toxicity, results from an imbalance between reactive oxygen species (ROS) generation and detoxification. As one consequence, protein carbonyl levels may become enhanced. Thus, the qualitative and quantitative description of protein carbonylation may be used to characterize how biological systems respond to oxidative stress induced by NPs. We investigated a representative panel of 24 NPs including functionalized amorphous silica (6), zirconium dioxide (4), silver (4), titanium dioxide (3), zinc oxide (2), multiwalled carbon nanotubes (3), barium sulfate and boehmite. Surface reactivities of all NPs were studied in a cell-free system by electron spin resonance (ESR). NRK-52E cells were treated with all NPs, analyzed for viability (WST-1 assay) and intracellular ROS production (DCFDA assay). Carbonylated proteins were assessed by 1D and/or 2D immunoblotting and identified by matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF). In parallel, tissue homogenates from rat lungs intratracheally instilled with silver NPs were studied. Eleven NPs induced elevated levels of carbonylated proteins. This was in good agreement with the surface reactivity of the NPs as obtained by ESR and the reduction in cell viability as assessed by WST-1 assay. By contrast, results obtained by DCFDA assay were deviating. Each NP induced an individual pattern of protein carbonyls on 2D immunoblots. Affected proteins comprised cytoskeletal components, proteins being involved in stress response, or cytoplasmic enzymes of central metabolic pathways such as glycolysis and gluconeogenesis. Furthermore, induction of carbonyls upon silver NP treatment was also verified in rat lung tissue homogenates. Analysis of protein carbonylation is a versatile and sensitive method to describe NP-induced oxidative stress and, therefore, can be used to identify NPs of concern. Furthermore, detailed information about compromised proteins may aid in classifying NPs according to their mode of action.

中文翻译：

---

蛋白质羰基化的蛋白质组学分析：揭示纳米颗粒毒性机制的有用工具。

氧化应激是解释纳米颗粒（NP）引起的毒性的常用方法，其原因是活性氧（ROS）生成和解毒之间的不平衡。结果，蛋白质羰基水平可能提高。因此，蛋白质羰基化的定性和定量描述可用于表征生物系统如何响应NPs诱导的氧化应激。我们研究了由24个NP组成的代表性面板，包括功能化的无定形二氧化硅（6），二氧化锆（4），银（4），二氧化钛（3），氧化锌（2），多壁碳纳米管（3），硫酸钡和勃姆石。在无细胞系统中，通过电子自旋共振（ESR）研究了所有NP的表面反应性。用所有NP处理NRK-52E细胞，分析生存力（WST-1测定）和细胞内ROS产生（DCFDA测定）。通过1D和/或2D免疫印迹评估羰基化的蛋白质，并通过基质辅助激光解吸飞行时间质谱（MALDI-TOF / TOF）进行鉴定。平行地，研究了气管内滴注了银纳米颗粒的大鼠肺组织匀浆。11个NP诱导了羰基化蛋白水平的升高。这与通过ESR获得的NPs的表面反应性以及通过WST-1分析评估的细胞活力的降低非常吻合。相比之下，通过DCFDA分析获得的结果是有偏差的。每个NP在2D免疫印迹上诱导蛋白质羰基的单独模式。受影响的蛋白质包括细胞骨架成分，参与应激反应的蛋白质，或主要代谢途径（例如糖酵解和糖异生）的细胞质酶。此外，在大鼠肺组织匀浆中也证实了银NP处理后对羰基的诱导。蛋白质羰基化分析是描述NP诱导的氧化应激的一种通用且灵敏的方法，因此可用于鉴定所关注的NP。此外，有关受损蛋白质的详细信息可能有助于根据作用方式对NP进行分类。