Large-scale proteomics analysis of the human kinome

Area of Science:

• Proteomics
• Cellular signaling
• Biochemistry

Background:

• Protein kinases regulate eukaryotic signal transduction pathways.
• Low abundance of protein kinases in proteome studies hinders comprehensive analysis.
• Enrichment strategies are needed for kinome-wide studies.

Purpose of the Study:

• To develop and evaluate methods for kinome-wide enrichment and analysis.
• To compare protein kinase expression across different cancer cell lines.
• To identify site-specific phosphorylation events in the human kinome.

Main Methods:

• Stable isotope labeling by amino acids in cell culture (SILAC) for quantitative comparison.
• Utilized kinase-selective affinity resins with pyrido[2,3-d]pyrimidine-based inhibitors.
• Employed a broadly selective kinase ligand (VI16832) for enrichment and quantitative mass spectrometry.

Main Results:

• Demonstrated feasibility of comparative kinome profiling using affinity resins.
• Quantified relative expression of over 170 protein kinases across three cancer cell lines.
• Identified distinct kinase expression patterns, with MV4-11 cells showing high cytoplasmic and low receptor tyrosine kinases.
• Revealed approximately 1200 phosphorylation sites on over 200 protein kinases.

Conclusions:

• Developed straightforward experimental procedures for kinase-selective proteomics.
• Comparative kinome profiling provides insights into signaling network architecture.
• Expanded the understanding of site-specific protein kinase regulation.
• Potential for future analysis of signal transduction and kinase drug targets.