RESUMO
Small-molecule fluorescent probes have been widely used in target identification, but this method has many disadvantages. For example, the identified proteins are usually complex, and additional biochemical studies are needed to distinguish real targets from interference results. To address this problem, we propose a series of strategies for improving the efficiency of target identification. First, pretreatment with a lower concentration of hydrogen peroxide can shield against thiol interference. Second, the use of benzophenone as a photo-affinity group is not appropriate, and diazirines are preferred. Third, if cytoskeleton proteins or stress proteins are captured, the interference must be carefully eliminated. The specificity of target identification can be improved by optimizing these three strategies. In this paper, we discuss the problems associated with the use of the click reaction in living cells and provide important complementary techniques for photo-affinity probes based on the click chemistry reaction.