Confirm compound engagement or identify ligandable cysteines
Reactive cysteine profiling is a powerful and flexible chemical proteomics tool designed to identify or confirm your drug’s binding site; identify off-target effects; or identify ligandable cysteines on a protein of interest.
A schematic of a reactive cysteine experiment is below.
Cells are treated with either a cysteine reactive compound or DMSO followed by cell lysis and incubation with the cysteine reactive probe, desthiobiotin-iodoacetamide (DBIA).
The DBIA probe with electrophilic functionality highlighted in yellow and the biotin enrichment handle in green.
Once incubated the with the cysteine reactive probe proteins are digested, free thiols are capped with iodoacetamide and peptides are labeled with Tandem Mass Tags and combined for multiplexed quantitative proteomics.
Cysteine reactive probe labeled peptides are enriched with streptavidin and analyzed with LC-MS/MS. Data are processed using FragPipe.
In the following example reactive cysteine profiling was used to identify the binding site of the CDK7 inhibitor THZ1. THZ1 is a covalent inhibitor of CDK7 with IC50 value of 3.2nM. THZ1 covalently modifies CDK7 by targeting the C312 residue outside of the kinase domain. This mechanism was reported in Jurkat cells by Kwiatkowski et al in a 2014 study titled “Targeting transcription regulation in cancer with a covalent CDK7 inhibitor”. We have repeated this observation in the human pancreatic cell line, AsPC-1.
We treated human pancreatic adenocarcinoma cells (AsPC-1) in triplicate with DMSO, 05µM and 5µM THZ1. Desthiobiotin-iodoacetamide (DBIA) was employed as a reactive cysteine probe. Following reduction with dithiothreitol free cysteines were alkylated with iodoacetamide. Samples were processed for multiplex quantitative proteomics using Tandem Mass TagsTM (TMT). Four-hour LC-MS/MS analysis was performed on a Waters M-Class HPLC interfaced to a ThermoFisher Exploris 480 mass spectrometer, and the data were processed using FragPipe.
Volcano plot showing differential analysis of the DMSO treated and 0.5µM THZ1 treated cells (n=3). The known THZ1 binding site is highlighted in blue.
Box plot for the C312 abundance with DMSO, 0.5µM and 5µM THZ1 treatment. The data show the decrease in the abundance of the site with treatment indicating increasing occupancy of the drug at that site.
This study highlights the value of reactive cysteine profiling for identifying drug binding sites. We have confirmed the CDK7 inhibitor THZ1 covalently modifies CDK7 at the C312 position in the AsPC-1 human pancreatic cancer cell line.
Gain Deeper Insights
This report explores how reactive cysteine profilin enables the selective identification and quantification of active proteins, providing critical data for drug discovery, biomarker identification, and enzyme acitivity analysis. Learn more about our methodologies and see how reactive cysteine profiling can enhance your research.
Learn More About How Advanced Mass Spectrometry Can Transform Your Research
Reach out and one of our scientists would be happy to discuss your project and how we can help.
How can we help?
Please click here to contact us