Analysis of recombinant phosphoprotein complexes with complementary mass spectrometry approaches

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• The baculovirus expression vector system is effective for producing complex recombinant proteins.
• Cyclin-dependent kinase (CDK)-activating kinase (CAK) is crucial for cell cycle regulation.
• Understanding CAK phosphorylation is vital for comprehending its function.

Purpose of the Study:

• To purify the recombinant CAK complex (CDK7, cyclin H, MAT1).
• To functionally characterize the purified CAK complex.
• To map phosphorylation sites and analyze heterogeneity using mass spectrometry.

Main Methods:

• Baculovirus expression vector system for protein production.
• In vitro kinase assays.
• Liquid chromatography-mass spectrometry (LC-MS) for intact protein analysis.
• Matrix-assisted laser desorption/ionization (MALDI)-MS and nanoLC-tandem mass spectrometry (MS/MS) for site mapping.

Main Results:

• Recombinant CAK complex was successfully purified.
• CAK phosphorylated CDK2 and RNA polymerase II CTD in vitro.
• Mass spectrometry revealed heterogeneous and incomplete phosphorylation across CAK subunits.
• Specific phosphorylation sites were identified on CDK7, cyclin H, and MAT1.

Conclusions:

• The recombinant CAK complex is functional and exhibits complex phosphorylation patterns.
• Mass spectrometry is a powerful tool for analyzing protein phosphorylation heterogeneity.
• Detailed phosphorylation site mapping provides insights into CAK regulation.