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Fig. 6 | Proteome Science

Fig. 6

From: Tetrazine ligation for chemical proteomics

Fig. 6

Target identification of Olaparib and Ibrutinib. a Structure of Olaparib target ID probe with diverse bioorthogonal groups. b Comparison of reaction efficiency of iEDDA, SPAAC, and CuAAC in affinity based enrichment (pull-down) assay. HuT78 nuclear lysate was incubates with target ID probes (3, 8, 9) and conjugated with biotin for 30 min (probe 3), 45 min (probe 8) and 90 min (probe 8 and 9). THPTA and sodium ascorbate were used for a ligand and a reducing agent of CuAAC. The target proteins were enriched using neutravidin beads, released from the beads and immunostained with anti-PARP1 antibody. The target protein enrichment efficiency was calculated based on quantification of PARP1 bands. c Comparison of reaction efficiency of iEDDA, SPAAC, and CuAAC in fluorescent cell imaging. 2 μM of target ID probes 3, 8, 9 or DMSO were treated to HeLa cells for 1 h, followed by fixation and permeablized. 3, 8, 9 were then conjugated with 100 nM TAMRA-Tz for 5 min, 10 μM Cy5.5-DBCO for 60 min and 10 μM TAMRA-azide for 60 min, respectively (scale bar: 25 μm). d Structure of Ibrutinib target ID probe functionalized with azide and TCO. e Comparison of target protein (BTK) specificity of SPAAC and iEDDA in fluorescent gel imaging. BV-173 lysate was treated with 11 and 12 in the presence or absence of Ibrutinib. Protein binding target ID probes were labeled with Cy5.5-DBCO or Cy5-Tz. Then the labeled proteins were visualized by SDS gel electrophoresis and fluorescence gel scanning. Reprinted with permission from ref 63. Copyright 2016 American Chemical Society

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