Protein-inhibitor complexes analyzed by alkaline capillary LC-MS.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• Liquid chromatography-mass spectrometry (LC-MS) is vital for determining protein molecular weights and analyzing protein-ligand complexes.
• Existing LC-MS methods have limitations, particularly for analyzing complexes unstable under certain pH conditions.

Purpose of the Study:

• To develop and validate robust LC-MS methods for accurate protein molecular weight determination.
• To investigate the binding modes of protein-ligand complexes, including those unstable under acidic conditions.
• To apply these methods to study inhibitors of CP88, a cysteine protease.

Main Methods:

• Development of small-bore and capillary LC-MS methods operating under both acidic and high pH conditions.
• Implementation of a high pH method crucial for analyzing acid-labile complexes.
• Establishment of a protocol for studying protein-ligand complexes under denaturing conditions.
• Application of the developed methods to analyze proprietary cysteine protease (CP88) inhibitors.

Main Results:

• Achieved microgram sensitivity for protein molecular weight measurements using both developed LC-MS methods.
• The high pH method enabled the study of complexes previously unstable under acidic conditions.
• Identified distinct binding modes for different CP88 inhibitors, despite similar non-reversible biochemical activity.
• Confirmed a reversible covalent binding mechanism for one inhibitor interacting with CP88.

Conclusions:

• The developed LC-MS methods provide sensitive and versatile tools for protein molecular weight analysis and the characterization of protein-ligand interactions.
• These methods are effective in elucidating complex binding behaviors, even under denaturing or pH-sensitive conditions.
• The study successfully differentiated inhibitor binding modes to CP88, offering insights into drug-target interactions.