Ibrutinib KiNativ -- single dose experiment

The KiNativ assay platform is based on the use of biotinylated acyl phosphates of ATP and ADP that act as probes by reacting with protein kinases on conserved lysine residues in the ATP binding pocket to covalently attach a biotin moiety. Using these probes, cell lysates treated with a kinase inhibitor can be labeled with BHAcATP or BHAcADP, with kinases inhibited by the compound of interest exhibiting reduced or no labeling. This is followed by digestion with trypsin, isolation of biotinylated peptides, and analysis by mass spectrometry to determine the extent of labeling of peptides from each kinase.

KiNativ检测平台以生物素酰化ATP酰基磷酸酯与ADP酰基磷酸酯为核心试剂：这类探针可与ATP结合口袋内保守赖氨酸残基上的蛋白激酶发生特异性反应，共价连接生物素基团。使用该类探针时，经激酶抑制剂处理的细胞裂解液可被BHAcATP或BHAcADP标记，而被目标化合物抑制的激酶的标记信号会出现减弱或完全缺失的情况。后续通过胰蛋白酶消化、生物素化肽段富集，以及质谱分析，即可测定每种激酶对应肽段的标记程度。