Chemical-peptide conjugation is the molecular initiating event in skin sensitization. The OECD test guideline uses a high-performance liquid chromatography/ultraviolet (HPLC/UV) detection method to quantify chemical-peptide conjugation in a direct peptide reactivity assay (DPRA), which measures the depletion of two synthetic peptides containing lysine or cysteine residues. To improve assay throughput, sensitivity and accuracy, an automated 384-well plate-based RapidFire solid-phase extraction (SPE) system coupled with tandem mass spectrometry (MS/MS) DPRA was developed and validated in the presence of newly designed internal standard. Compared to the HPLC/UV-based DPRA, the automated SPE-MS/MS-based DPRA improved throughput from 16 minutes to 10 seconds per sample, and substrate peptides usage was reduced from 100 mM to 5 µM. When implementing the SPE-MS/MS-based DPRA into a high-throughput platform, we found 10 compounds depleting lysine peptide and 23 compounds depleting cysteine peptide (including 5 unreported chemicals) in a concentration-response manner. The adduct formation between cysteine and cinnamic aldehyde and ethylene glycol dimethacrylate were further analyzed using high-performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF-MS) to confirm the conjugation. Overall, the automated SPE-MS/MS-based platform is an efficient, economic, and accurate way to detect skin sensitizers.

中文翻译：

---

使用高通量质谱筛选平台检测皮肤致敏剂的直接肽反应性测定

化学肽结合是皮肤致敏的分子起始事件。OECD 测试指南使用高效液相色谱/紫外线 (HPLC/UV) 检测方法来量化直接肽反应性测定 (DPRA) 中的化学肽缀合，该测定可测量含有赖氨酸或半胱氨酸残基的两种合成肽的消耗量。为了提高分析通量、灵敏度和准确性，开发了一种基于 384 孔板的自动化 RapidFire 固相萃取 (SPE) 系统与串联质谱 (MS/MS) DPRA 相结合，并在新设计的内标存在下进行了验证。与基于 HPLC/UV 的 DPRA 相比，基于 SPE-MS/MS 的自动化 DPRA 将每个样品的通量从 16 分钟提高到 10 秒，底物肽的使用量从 100 mM 减少到 5 µM。在将基于 SPE-MS/MS 的 DPRA 实施到高通量平台时，我们以浓度响应方式发现了 10 种消耗赖氨酸肽的化合物和 23 种消耗半胱氨酸肽的化合物（包括 5 种未报告的化学物质）。使用高效液相色谱飞行时间质谱 (HPLC-TOF-MS) 进一步分析半胱氨酸与肉桂醛和乙二醇二甲基丙烯酸酯之间的加合物形成，以确认共轭。总体而言，基于 SPE-MS/MS 的自动化平台是一种高效、经济且准确的皮肤致敏剂检测方法。使用高效液相色谱飞行时间质谱 (HPLC-TOF-MS) 进一步分析半胱氨酸与肉桂醛和乙二醇二甲基丙烯酸酯之间的加合物形成，以确认共轭。总体而言，基于 SPE-MS/MS 的自动化平台是一种高效、经济且准确的皮肤致敏剂检测方法。使用高效液相色谱飞行时间质谱 (HPLC-TOF-MS) 进一步分析半胱氨酸与肉桂醛和乙二醇二甲基丙烯酸酯之间的加合物形成，以确认共轭。总体而言，基于 SPE-MS/MS 的自动化平台是一种高效、经济且准确的皮肤致敏剂检测方法。