Ultradeep Phosphoproteomics for Assessing Protein Kinase Inhibitor Selectivity on a Proteome Scale.

Area of Science:

• Biochemistry
• Proteomics
• Pharmacology

Background:

• Protein kinase inhibitors (PKIs) selectivity is crucial for effective drug discovery.
• Understanding PKI mechanisms of action requires precise selectivity profiling.

Purpose of the Study:

• To develop and validate an ultradeep phosphoproteomics approach for assessing PKI selectivity.
• To elucidate the mechanism of action of Zanubrutinib using this novel method.

Main Methods:

• Utilized two complementary phosphoproteomics strategies: Zr4+-IMAC/TiO2 enrichment with DIA and SH2-Superbinder enrichment with DDA.
• Applied bioinformatics analysis to identify significantly altered phosphosites upon Zanubrutinib stimulation.

Main Results:

• Identified 97 and 316 significantly altered phosphosites in DDA and DIA datasets, respectively.
• Generated a detailed selectivity profile for Zanubrutinib, revealing molecular-level insights into its mechanism.

Conclusions:

• The developed ultradeep phosphoproteomics approach is more comprehensive, high-throughput, and precise than existing methods.
• This method advances PKI selectivity assessment and broadens cell signaling research capabilities.