Quantitative Characterization of Three Carbonic Anhydrase Inhibitors by LESA Mass Spectrometry

Area of Science:

• Analytical Chemistry
• Biochemistry
• Biophysics

Background:

• Liquid extraction surface analysis (LESA) coupled with native mass spectrometry (MS) offers sensitive, rapid, and automatable analytical capabilities.
• Quantitative analysis of protein-ligand interactions is crucial for understanding biological processes and drug discovery.

Purpose of the Study:

• To evaluate the suitability of LESA-MS for quantitatively determining equilibrium dissociation constants (Kd) of protein-ligand interactions.
• To compare LESA-MS quantitative results with established methods like direct infusion MS and surface plasmon resonance (SPR).

Main Methods:

• Native LESA-MS was employed to analyze interactions between bovine carbonic anhydrase II and three known ligands: chlorothiazide, dansylamide, and sulfanilamide.
• Two LESA sampling strategies were investigated: premixing protein and ligand before deposition, and depositing protein followed by ligand in the sampling solvent.
• Kd values were calculated and compared across different methods and LESA approaches.

Main Results:

• LESA-MS provided consistent and accurate Kd values when the protein and ligand were premixed in solution prior to sample deposition.
• Inconsistent Kd values were obtained when the ligand was introduced only in the LESA sampling solvent.
• LESA-MS results showed good agreement with direct infusion MS and SPR when the premixed sample approach was used.

Conclusions:

• LESA-MS is a viable technique for the quantitative analysis of protein-ligand interactions.
• Accurate determination of Kd values using LESA-MS requires careful sample preparation, specifically premixing of protein and ligand before analysis.
• This method holds promise for high-throughput screening and characterization of molecular interactions.