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Updated: Oct 21, 2025
An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
Published on: June 6, 2022
PhosphoFlowSeq - A High-throughput Kinase Activity Assay for Screening Drug Resistance Mutations in EGFR
Anja Wagner1, Magdalena Teufl2, Lukas Gold2
1Department of Chemistry, Institute of Biochemistry, University of Natural Resources and Life Sciences, Vienna, Austria; Department of Biotechnology, Institute of Molecular Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
Abstract:
Drug resistance poses a major challenge for targeted cancer therapy. To be able to functionally screen large randomly mutated target gene libraries for drug resistance mutations, we developed a biochemically defined high-throughput assay termed PhosphoFlowSeq. Instead of selecting for proliferation or resistance to apoptosis, PhosphoFlowSeq directly analyzes the enzymatic activities of randomly mutated kinases, thereby reducing the dependency on the signaling network in the host cell. Moreover, simultaneous analysis of expression levels enables compensation for expression-based biases on a single cell level. Using EGFR and its kinase inhibitor erlotinib as a model system, we demonstrate that the clinically most relevant resistance mutation T790M is reproducibly detected at high frequencies after four independent PhosphoFlowSeq selection experiments. Moreover, upon decreasing the selection pressure, also mutations which only confer weak resistance were identified, including T854A and L792H. We expect that PhosphoFlowSeq will be a valuable tool for the prediction and functional screening of drug resistance mutations in kinases.
Insights
PhosphoFlowSeq is a new assay that directly measures kinase enzymatic activity to find cancer drug resistance mutations. This method successfully identified known and novel resistance mutations in EGFR, aiding future drug discovery.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Drug resistance is a significant hurdle in targeted cancer therapy.
- Existing screening methods often rely on indirect measures like cell proliferation or apoptosis.
Purpose of the Study:
- To develop a high-throughput assay, PhosphoFlowSeq, for direct functional screening of drug resistance mutations in kinases.
- To overcome limitations of current methods by analyzing enzymatic activity and expression levels simultaneously.
Main Methods:
- Developed PhosphoFlowSeq, a biochemically defined high-throughput assay.
- Applied the assay to a model system using EGFR (Epidermal Growth Factor Receptor) and erlotinib.
- Performed functional screening of randomly mutated kinase libraries.
Main Results:
- PhosphoFlowSeq reproducibly detected the clinically relevant EGFR resistance mutation T790M.
- Identified weaker resistance mutations (T854A, L792H) by adjusting selection pressure.
- Demonstrated simultaneous analysis of kinase activity and expression levels for accurate bias compensation.
Conclusions:
- PhosphoFlowSeq enables direct functional screening of kinase drug resistance mutations.
- The assay is valuable for identifying both major and minor resistance mutations.
- PhosphoFlowSeq is expected to advance the prediction and screening of kinase drug resistance mutations.
