Characterization of human alcohol dehydrogenase isoenzymes by capillary isoelectric focusing-mass spectrometry

The human liver alcohol dehydrogenase (ADH) isoenzymes are currently believ ed to play a major role in ethanol metabolism, accounting for most of the e thanol oxidized in the liver. They have similar molecular masses and simila r isoelectric point (pi) values (the 13 possible isoenzymes having pls in t he range of 8.26-8.87), making their characterization a significant analyti cal challenge. Capillary isoelectric focusing (CIEF) coupled on-line with e lectrospray ionization - Fourier transform ion cyclotron resonance (ESI-FTI CR) mass spectrometry was applied to separate and characterize mixtures of alpha alpha, beta(1)beta(1) and beta(3)beta(3) ADH isoenzymes. Seven differ ent species were resolved by the separation in the p/ 8.26-8.67 range. ESI- FTICR analysis of native ADHs revealed that each noncovalent ADH complex co ntains two monomeric protein units and four zinc atoms. The combination of CIEF separations with mass spectrometry appears well-suited for detailed ch aracterization of ADH isozymes, and the attomole level sensitivity of FTICR should allow very small samples to be addressed.