Investigating\nActive Site Binding of Ligands to High\nand Low Activity Carbonic Anhydrase Enzymes Using Native Mass Spectrometry

Carbonic anhydrases (CAs) are a family of enzymes that\nplay an\nimportant pH regulatory role in health and disease. While different\nCA isozymes have a high degree of structural similarity, they have\nvariable enzymatic activity, with CA III being the least active and\nhaving less than 1% of the activity of CA II, the most active. Furthermore,\nligand binding studies for CA III are limited, and a resulting lack\nof chemical probes impedes understanding of this CA isozyme in comparison\nto other CA family members where studies are abundant. Therefore,\nwe employed native mass spectrometry (nMS), also known as intact mass\nspectrometry, to assess ligand binding to CA II and CA III and discovered\ntwo novel compounds that for the first time display strong binding\nto CA III. We present a new data visualization and quantification\ntool developed to display native mass spectra as an intuitive stacked\nheat map representation for rapidly interpreting the results of ligand-protein\nbinding from nMS screening.