Quantitative Characterization of Three Carbonic Anhydrase Inhibitors by LESA Mass Spectrometry

Liquid
extraction surface analysis (LESA) coupled to native mass
spectrometry (MS) presents unique analytical opportunities due to
its sensitivity, speed, and automation. Here, we examine whether this
tool can be used to quantitatively probe protein–ligand interactions
through calculation of equilibrium dissociation constants (Kd values). We performed native LESA MS analyses
for a well-characterized system comprising bovine carbonic anhydrase
II and the ligands chlorothiazide, dansylamide, and sulfanilamide,
and compared the results with those obtained from direct infusion
mass spectrometry and surface plasmon resonance measurements. Two
LESA approaches were considered: In one approach, the protein and
ligand were premixed in solution before being deposited and dried
onto a solid substrate for LESA sampling, and in the second, the protein
alone was dried onto the substrate and the ligand was included in
the LESA sampling solvent. Good agreement was found between the Kd values derived from direct infusion MS and
LESA MS when the protein and ligand were premixed; however, Kd values determined from LESA MS measurements
where the ligand was in the sampling solvent were inconsistent. Our
results suggest that LESA MS is a suitable tool for quantitative analysis
of protein–ligand interactions when the dried sample comprises
both protein and ligand.