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Technical note
Chemoproteomics

Covalent inhibitor profiling

Unbiased, proteome-wide profiling of the cellular targets of cysteine-directed covalent inhibitors.

Covalent small molecule inhibitors can offer greater potency and longer-lasting effects than reversible drugs. Afatinib (EGFR), ibrutinib (BTK), neratinib (ErbB) and ARS-853 (KRAS G12C) are examples. Our unbiased LC-MS assay screens for the targets of cysteine-directed covalent inhibitors across the whole proteome.

At a glance

  • About 20,000 reactive cysteine sites quantified per experiment
  • High-efficiency streptavidin-based enrichment
  • TMT 18-plex with RTS-MS3 on an Orbitrap Eclipse
  • Suited to target identification, selectivity profiling and high-throughput screening

How it works

Whole-cell lysates are treated with a titration series of a cysteine-specific covalent inhibitor, in this example FMK. All unreacted cysteines are then capped with desthiobiotin-iodoacetamide. The proteomes are digested, labeled with TMT and mixed, and the reactive cysteine peptides are enriched on streptavidin and quantified by LC-MS. Drug targets show a characteristic loss of signal as the drug concentration rises.

Schematic of the competitive cysteine profiling workflow, from inhibitor titration through TMT barcoding, streptavidin enrichment and LC-MS quantification.
Competitive cysteine profiling: lysates are treated with an inhibitor titration, unreacted cysteines are capped with desthiobiotin-iodoacetamide, then samples are digested, TMT-labeled, enriched on streptavidin and quantified by LC-MS.

Example data: FMK selectivity

Comparing the 200 nM FMK treatment with the DMSO control shows the inhibitor's specificity: a peptide in the active site of its known target, RPS6KA1, is selectively depleted against a background of more than 8,000 quantified cysteine peptides.

Volcano plot of cysteine peptide fold change versus significance at 200 nM FMK, with two RPS6KA1 active-site peptides standing out as strongly depleted.
At 200 nM, FMK selectively depletes a cysteine peptide in the active site of its known target RPS6KA1, against a background of more than 8,000 quantified peptides.

Spotlight: the RPS6KA1 active site

Across the full FMK titration, run in duplicate, the RPS6KA1 active-site peptide shows close agreement between replicates and a sharp, dose-dependent loss of signal at the highest concentrations.

Bar chart of the RPS6KA1 peptide ETIGVGSYSECK across an FMK titration from DMSO to 200 nM, falling to about 15% of control at 200 nM.
The RPS6KA1 active-site peptide drops sharply at the highest FMK dose, with close agreement between duplicates.

For the full covalent inhibitor workflow, including targeted and direct engagement assays, see our Chemoproteomics service page.

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