Activity-based ubiquitin probes (Ub-ABPs) have recently been developed as effective tools for studying the capabilities of E1–E2–E3 enzymes, but most of them can only be used in cell lysates. Here, we report the first cell-penetrating Ub-Dha probes based on thiazolidine-protected cysteines, which enable successful delivery into cells confirmed by a fluorophore at the N-terminus of Ub and live-cell fluorescence microscopy. A total of 18 E1–E2–E3 enzymes in live cells were labelled and enriched in combination with label-free quantification (LFQ) mass spectrometry. This work provided a new cell-penetrating Ub tool for studying the activity and function of Ub-related enzymes.

中文翻译：

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一种可渗透细胞的 Ub-Dha 探针，用于分析活细胞中的 E1–E2–E3 酶

基于活性的泛素探针 (Ub-ABP) 最近被开发为研究 E1-E2-E3 酶功能的有效工具，但大多数只能用于细胞裂解物。在这里，我们报告了第一个基于噻唑烷保护的半胱氨酸的细胞穿透 Ub-Dha 探针，该探针能够成功递送到由 Ub N 末端的荧光团和活细胞荧光显微镜证实的细胞中。结合无标记定量 (LFQ) 质谱法对活细胞中总共 18 种 E1–E2–E3 酶进行标记和富集。这项工作为研究Ub相关酶的活性和功能提供了一种新的细胞穿透Ub工具。