BCMB Allied Program, Weill Cornell Medical College, Cornell University, New York, New York.
Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York.
Curr Protoc. 2021 Aug;1(8):e213. doi: 10.1002/cpz1.213.
Protein methyltransferases (PMTs) regulate many aspects of normal and disease processes through substrate methylation, with S-adenosyl-L-methionine (SAM) as a cofactor. It has been challenging to elucidate cellular protein lysine and arginine methylation because these modifications barely alter physical properties of target proteins and often are context dependent, transient, and substoichiometric. To reveal bona fide methylation events associated with specific PMT activities in native contexts, we developed the live-cell Bioorthogonal Profiling of Protein Methylation (lcBPPM) technology, in which the substrates of specific PMTs are labeled by engineered PMTs inside living cells, with in situ-synthesized SAM analogues as cofactors. The biorthogonality of this technology is achieved because these SAM analogue cofactors can only be processed by the engineered PMTs-and not native PMTs-to modify the substrates with distinct chemical groups. Here, we describe the latest lcBPPM protocol and its application to reveal proteome-wide methylation and validate specific methylation events. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Live-cell labeling of substrates of protein methyltransferases GLP1 and PRMT1 with lcBPPM-feasible enzymes and SAM analogue precursors Support Protocol: Gram-scale synthesis of Hey-Met Basic Protocol 2: Click labeling of lcBPPM cell lysates with a biotin-azide probe Alternate Protocol: Click labeling of small-scale lcBPPM cell lysates with a TAMRA-azide dye for in-gel fluorescence visualization Basic Protocol 3: Enrichment of biotinylated lcBPPM proteome with streptavidin beads Basic Protocol 4: Proteome-wide identification of lcBPPM targets with mass spectrometry Basic Protocol 5: Validation of individual lcBPPM targets by western blot.
蛋白质甲基转移酶（PMTs）通过底物甲基化来调节正常和疾病过程的许多方面，其中 S-腺苷甲硫氨酸（SAM）作为辅助因子。由于这些修饰几乎不会改变靶蛋白的物理性质，并且常常依赖于上下文、短暂和亚化学计量，因此阐明细胞蛋白赖氨酸和精氨酸甲基化一直具有挑战性。为了在天然环境中揭示与特定 PMT 活性相关的真正甲基化事件，我们开发了活细胞蛋白甲基化的生物正交分析（lcBPPM）技术，其中特定 PMT 的底物在活细胞内由工程 PMT 标记，原位合成的 SAM 类似物作为辅助因子。该技术的生物正交性是因为这些 SAM 类似物辅助因子只能被工程 PMT 而不是天然 PMT 处理，以用不同的化学基团修饰底物。在这里，我们描述了最新的 lcBPPM 方案及其在揭示全蛋白质组甲基化和验证特定甲基化事件中的应用。© 2021 Wiley Periodicals LLC。基本方案 1：用 lcBPPM 可行的酶和 SAM 类似物前体对蛋白质甲基转移酶 GLP1 和 PRMT1 的底物进行活细胞标记支持方案：Hey-Met 的克级合成基本方案 2：用生物素-叠氮探针对 lcBPPM 细胞裂解物进行点击标记备选方案：用 TAMRA-叠氮染料对小范围 lcBPPM 细胞裂解物进行点击标记，用于胶内荧光可视化基本方案 3：用链霉亲和素珠富集生物素化的 lcBPPM 蛋白质组基本方案 4：用质谱法对 lcBPPM 靶标进行全蛋白质组鉴定基本方案 5：用 western blot 验证单个 lcBPPM 靶标。
