荧光标记在蛋白质识别中起到了重要作用。然而，目前大多数基于Michael反应的荧光标记均是不可逆的。在标记过程中，它们不但会对蛋白质的构象与活性带来不利影响；而且难以实现与被标记蛋白质的分离。针对这些问题，在前期研究中我们报道了基于可逆策略的GSH的定量分析方法。在此基础上，作为后续深入研究，本项目提出了基于“可逆Michael反应”机理的蛋白质荧光标记方法。该方法不仅能实现对蛋白质巯基特异性的标记，而且可在温和条件下实现荧光探针与目标蛋白质的分离，实现对荧光标记过程的“精确可控”。我们期望该策略将为深入研究蛋白质的识别提供坚实的基础。

Fluorescent labeling techniques play an essential role in identification of proteins. However, most fluorescent probe that based on the Michael addition are irreversible, making them not suitable for this purpose due to that they might denature the conformation of the proteins during labeling processes. On the other hand, these probes are also demonstrated to be with difficult in separation from the labeled proteins. To this end, we proposed previously a reversible strategy for quantitating nonprotein thiols. As a further research, we presented here a method, based on a "reversible Michael addition reaction" mechanism, for protein labeling. The method, not only make the labeling of protein more effectively, but also facilitate the separation of the fluorescent probe from target proteins under a mild condition. Therefore, this method proposed here could provide a powerful and reliable tool for us to further study protein recognitions. We hope that the novel idea proposed in this proposal may stimulate this rapidly expanding multidisciplinary research field.