Recently, numerous efforts have been devoted to identifying drug targets and binding sites in complex proteomes, which is of great importance in modern drug discovery. In this study, we developed a robust lysine reactivity profiling method to systematically study drug-binding targets and binding sites at the proteome level. This method is based on the principle that binding of a drug to a specific region of target proteins will change the reactivity of lysine residues that are located at this region, and these changes can be detected with an enrichable and lysine reactive probe. Coupled with data-independent acquisition (DIA), the known target proteins and corresponding binding sites were successfully revealed from K562 cell lysates for three model drugs: geldanamycin, staurosporine, and dasatinib. In addition, the druginduced conformational changes of certain targets were also revealed by our method during the screening of staurosporine. The screening sensitivity of our method revealed from the screening of stuarosporine and dasatinib was comparable with that of thermal proteome profiling (TPP) or machine learning-based limited proteolysis (LiP-Quant). Overall, 21 and 4 kinase targets, including adenosine 5′-triphosphate (ATP)-binding targets, were identified for staurosporine and dasatinib in K562 cell lysates, respectively. We found that target proteins identified by TPP, LiP-Quant, and our method were complementary, emphasizing that the development of new methods that probe different properties of proteins is of great importance in drug target deconvolution. We also envision further applications of our method in proteome-wide probing multiple events that involve lysine reactivity changes.
Protein arginine methylation plays an important role in regulating protein functions in different cellular processes, and its dysregulation may lead to a variety of human diseases. Recently, arginine methylation was found to be involved in modulating protein liquid–liquid phase separation (LLPS), which drives the formation of different membraneless organelles (MLOs). Here, we developed a steric effect–based chemical-enrichment method (SECEM) coupled with liquid chromatography–tandem mass spectrometry to analyze arginine dimethylation (DMA) at the proteome level. We revealed by SECEM that, in mammalian cells, the DMA sites occurring in the RG/RGG motifs are preferentially enriched within the proteins identified in different MLOs, especially stress granules (SGs). Notably, global decrease of protein arginine methylation severely impairs the dynamic assembly and disassembly of SGs. By further profiling the dynamic change of DMA upon SG formation by SECEM, we identified that the most dramatic change of DMA occurs at multiple sites of RG/RGG–rich regions from several key SG-contained proteins, including G3BP1, FUS, hnRNPA1, and KHDRBS1. Moreover, both in vitro arginine methylation and mutation of the identified DMA sites significantly impair LLPS capability of the four different RG/RGG–rich regions. Overall, we provide a global profiling of the dynamic changes of protein DMA in the mammalian cells under different stress conditions by SECEM and reveal the important role of DMA in regulating protein LLPS and SG dynamics.
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