Label-Free Quantitative Proteomics and Substrate-Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in Ex Vivo Human Skin and a Human Living Skin Equivalent Model
Label-Free Quantitative Proteomics and Substrate-Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in Ex Vivo Human Skin and a Human Living Skin Equivalent Model
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DOI：
10.1124/dmd.120.000168
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发表时间：
2021-01-01
影响因子：
3.9
通讯作者：
Barber, Jill
中科院分区：
文献类型：
--
作者：
Couto, Narciso;Newton, Jillian R. A.;Barber, Jill
我们报告的第一次无标记的定量异生素代谢酶（XME），转运蛋白，氧化还原酶，蛋白酶，和核酸酶在六个人的皮肤外植体和一个三维的生活皮肤等效模型从LabSkin。我们的目的是评估LabSkin作为替代动物试验的局部制剂开发的适用性。从总细胞蛋白中鉴定和定量了2000多种蛋白质。乙醇脱氢酶1C，人类皮肤中最丰富的I相XME，和谷胱甘肽S-转移酶pi 1，人类皮肤中最丰富的II相XME，在LabSkin中以相似的丰度存在。对几种酯酶进行定量，并使用基于底物的质谱成像在LabSkin中确认酯酶活性。对于测试的底物，没有观察到细胞色素P450（P450）活性，这与蛋白质组学数据一致，其中同源P450在人皮肤和LabSkin中都不存在。无标记蛋白质定量允许洞察其他相关过程，如氧化还原稳态和蛋白质水解。例如，最丰富的抗氧化酶是硫氧还蛋白和过氧化物氧还蛋白-1。该系统性测定人类皮肤和LabSkin之间的功能等效性是构建具有代表性的人类体外皮肤模型的关键一步，该模型可用作当前基于动物的化学安全性测试的替代方法，并用于预测局部给药药物的剂量。人皮肤中的氧化还原酶、蛋白酶和核酸酶增强了我们对皮肤生理学和局部药物和化妆品的生物转化的理解。这将有助于开发数学模型来预测人类皮肤中的药物代谢，并开发更强大的体外工程人类皮肤组织作为动物试验的替代品。
We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases, and nucleases in six human skin explants and a three-dimensional living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C, the most abundant phase I XME in human skin, and glutathione S-transferase pi 1, the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (P450) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate P450s were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin and peroxiredoxin-1. This systematic determination of functional equivalence between human skin and LabSkin is a key step toward the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs.SIGNIFICANCE STATEMENTThe use of label-free quantitative mass spectrometry to elucidate the abundance of xenobiotic metabolizing enzymes, transporters, redox enzymes, proteases, and nucleases in human skin enhance our understanding of the skin physiology and biotransformation of topical drugs and cosmetics. This will help to develop mathematical models to predict drug metabolism in human skin and to develop more robust in vitro engineered human skin tissue as alternatives to animal testing.