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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Nat Chem Biol. 2022 May 19;18(9):934–941. doi: 10.1038/s41589-022-01019-1

Figure 2.

Figure 2.

Salicylaldehyde probes exhibit prolonged kinase residence time in cells. (a) Salicylaldehyde probes 3 and 4. (b,c) Jurkat cells were treated with 3 (b) or 4 (c) (2 μM, 30 min), followed by compound washout for the indicated times and lysis in the presence of sodium borohydride. After click conjugation with TAMRA-azide, samples were analyzed by in-gel fluorescence and Coomassie blue staining (Supplementary Figure 2). Data are representative of two independent experiments. (d-f) Jurkat cells were treated with DMSO or probes 1, 3, and 4 (2 μM, 30 min), followed by washout as described in the main text. After click conjugation with biotin-azide, modified proteins were enriched with Neutravidin-agarose and digested on-bead with trypsin. Enriched peptides were analyzed by LC-MS/MS and label-free quantitation (LFQ). (d) Shared and unique kinases enriched by each probe. (e) Median intensity values (LFQ) for identified kinases and non-kinases (mean ± SD, n=3). (f) Heat map showing estimated half-lives (t1/2) of kinases enriched from cells treated with probes 3 and 4, followed by washout for 0, 1, 3, and 6 hr. Inset depicts kinases with prolonged engagement post-washout. Grey bars (N.D.): kinase not detected or did not meet criteria for t1/2 determination (see main text).