Extended Data Fig. 8: Leveraging tryptic peptide maps from protein-directed ABPP experiments to deduce stereoselectively liganded residues.
From: Multi-tiered chemical proteomic maps of tryptoline acrylamide–protein interactions in cancer cells
a, Sequence of C15orf57 showing the three cysteines (yellow highlight), peptides that were quantified in protein-directed ABPP (red), and the tryptic peptide containing the liganded cysteine, C111 (underlined). b, Competitive gel-ABPP data showing stereoselective blockade of WX-01-12 (5 µM, 1 h) engagement of C15orf57 by WX-02-46 (20 µM, 1 h pre-treatment). c, Competitive gel-ABPP data showing concentration-dependent, enantioselective blockade of WX-03-346 (1 µM, 1 h) engagement of STK39 by WX-03-59 (1 h pre-treatment) (left) and quantitation of these data (right). d, e, Crystal structure of STK39 (PDB: 5D9H) showing location of stereoprobe-liganded cysteine C334 (red) distal to the ATP pocket and highlighted in yellow are residues around C334 ( < 15 Å) that are conserved between STK39 and paralog OXSR1 (yellow-highlighted residues also shown in the sequence alignment in e). f, Protein-directed ABPP data showing stereoselective enrichment of AK3 by WX-03-338 and blockade of this enrichment by WX-03-57 in 22Rv1 cells. Data represent mean values ± s.d., n = 4 biological replicates. g, Tryptic peptide map of AK3 from protein-directed ABPP experiments showing stereoselective enrichment of all quantified AK3 peptides by WX-03-338 except for the peptide containing K34 (red). In the heat map display, tryptic peptide signal intensities were normalized to 100% for the WX-03-338 treatment group. h, Left, cysteine-directed ABPP data showing stereoselective liganding of FOXA1_C258 by WX-02-26 in 22Rv1 cells. Right, protein-directed ABPP data showing stereoselective enrichment of FOXA1 by WX-01-02 and blockade of this enrichment by WX-02-26 in 22Rv1 cells. Data represent mean values ± s.d., n = 4 biological replicates. i, Tryptic peptide map of FOXA1 from protein-directed ABPP experiments showing stereoselective enrichment of all quantified FOXA1 peptides by WX-01-02 except for the peptide containing C258 (red). In the heat map display, tryptic peptide signal intensities were normalized to 100% for the WX-01-02 treatment group. j, Gel-ABPP data demonstrating stereoselective engagement of recombinant WT-AK3, but not the K34R-AK3 mutant by WX-01-05. Experiments were performed in transfected HEK293T cells as described in Fig. 4b. k, Lysine-directed ABPP showing stereoselective liganding of AK3_K34 by WX-03-57. Data represent mean and individual values from two biological replicates. l, Crystal structure of AK3 (PDB: 6ZJD) showing distal location of K34 relative to the enzyme active site. For b, c, and j, data are from a single experiment representative of at least two independent experiments; IB = anti-Flag immunoblot, UT= untransfected cells.
