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. Author manuscript; available in PMC: 2013 Jul 1.
Published in final edited form as: Nat Chem Biol. 2012 Nov 11;9(1):43–50. doi: 10.1038/nchembio.1118

Figure 1. An active-site directed probe for ratiometric profiling of protein kinases.

Figure 1

(a) The cancer drug dasatinib in complex with the tyrosine kinase SRC (PDB code 3G5D). The arrow shows the site where dasatinib was modified with a benzophenone photo-crosslinker and an orthogonal chemical tag. (b) The chemical structure of probe 1. Probe 1 contains three components: (i) a potent ATP-competitive inhibitor (dasatanib), (ii) a photo-reactive benzophenone crosslinker, and (iii) a hexylchloride tag that selectively labels the active site of the self-labeling protein HaloTag. (c) Experimental crosslinking schematic using 1. Prior to photo-crosslinking experiments, HaloTag is labeled with 1. HT-1 is incubated with a kinase target and then irradiated with UV light. (d). HT-1 efficiently labels the recombinant SRC-family kinases (SFKs) SRC and HCK in cell lysate. Purified SRC or HCK (100 nM) was photo-crosslinked with HT-1 in mammalian cell lysate. Immunoblotting with an anti-SFK antibody shows that a large percentage of SRC and HCK are covalently modified. Upon addition of a dasatinib competitor, no mass-shifted kinases are observed.