LC-MS-Based Characterization of the Peptide Reactivity of Chemicals to Improve the In Vitro Prediction of the Skin Sensitization Potential
LC-MS-Based Characterization of the Peptide Reactivity of Chemicals to Improve the In Vitro Prediction of the Skin Sensitization Potential
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DOI：
10.1093/toxsci/kfn194
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发表时间：
2008-12-01
影响因子：
3.8
通讯作者：
Gfeller, Hans
中科院分区：
文献类型：
--
作者：
Natsch, Andreas;Gfeller, Hans
皮肤致敏过程的关键步骤是皮肤致敏剂与皮肤中的内源性蛋白质和/或肽之间形成共价加合物。基于这种机制的理解，人们对体外测定重新产生了兴趣，以确定化学物质对肽的反应性，以预测其致敏潜力。标准化的肽反应性测定产生了有希望的预测性。该已发表的测定方法基于带有紫外线检测的高效液相色谱法，以量化与测试化学品一起孵育后的肽消耗。我们观察到肽耗尽可能是由于加合物形成或肽氧化所致。在这里，我们报告了一种基于液相色谱-质谱（LC-MS）分析和游离硫醇基团检测的改进测定法。这种方法可以同时测定 (1) 肽消耗、(2) 肽氧化（二聚化）、(3) 加合物形成和 (4) 硫醇反应性，从而生成分子反应性的更详细表征。通过动力学测量进一步区分高反应性分子。该测定已针对 80 种化学品进行了验证。通过 LC-MS 检测和硫醇基团的消耗可以准确地定量肽消耗。大多数中度/强/极度敏化剂形成可检测的肽加合物，但许多敏化剂也能够催化肽氧化。虽然仅在敏化剂中观察到加合物形成，但在两种非敏化香料醛中也观察到了这种氧化反应，这表明肽耗尽可能并不总是被视为将化学品评级为敏化剂的充分证据。因此，这种改进的测定可以更全面地了解化学品的肽反应性，以更好地预测其致敏潜力。
A key step in the skin sensitization process is the formation of a covalent adduct between skin sensitizers and endogenous proteins and/or peptides in the skin. Based on this mechanistic understanding, there is a renewed interest in in vitro assays to determine the reactivity of chemicals toward peptides in order to predict their sensitization potential. A standardized peptide reactivity assay yielded a promising predictivity. This published assay is based on high-performance liquid chromatography with ultraviolet detection to quantify peptide depletion after incubation with test chemicals. We had observed that peptide depletion may be due to either adduct formation or peptide oxidation. Here we report a modified assay based on both liquid chromatography-mass spectrometry (LC-MS) analysis and detection of free thiol groups. This approach allows simultaneous determination of (1) peptide depletion, (2) peptide oxidation (dimerization), (3) adduct formation, and (4) thiol reactivity and thus generates a more detailed characterization of the reactivity of a molecule. Highly reactive molecules are further discriminated with a kinetic measure. The assay was validated on 80 chemicals. Peptide depletion could accurately be quantified both with LC-MS detection and depletion of thiol groups. The majority of the moderate/strong/extreme sensitizers formed detectable peptide adducts, but many sensitizers were also able to catalyze peptide oxidation. Whereas adduct formation was only observed for sensitizers, this oxidation reaction was also observed for two nonsensitizing fragrance aldehydes, indicating that peptide depletion might not always be regarded as sufficient evidence for rating a chemical as a sensitizer. Thus, this modified assay gives a more informed view of the peptide reactivity of chemicals to better predict their sensitization potential.