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

Abstract

The human liver alcohol dehydrogenase (ADH) isoenzymes are currently believed to play a major role in ethanol metabolism, accounting for most of the ethanol oxidized in the liver. They have similar molecular masses and similar isoelectric point (pI) values (the 13 possible isoenzymes having pIs in the range of 8.26-8.87), making their characterization a significant analytical challenge. Capillary isoelectric focusing (CIEF) coupled on-line with electrospray ionization - Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry was applied to separate and characterize mixtures of alphaalpha, beta1beta1 and beta3beta3 ADH isoenzymes. Seven different species were resolved by the separation in the pI 8.26-8.67 range. ESI-FTICR analysis of native ADHs revealed that each noncovalent ADH complex contains two monomeric protein units and four zinc atoms. The combination of CIEF separations with mass spectrometry appears well-suited for detailed characterization of ADH isozymes, and the attomole level sensitivity of FTICR should allow very small samples to be addressed.

Capillary electrophoresis of proteins 1999-2001

“…A dynamic CIEF simulator has been developed to model separation of up to 150 components [124]. CIEF has been used to analyze cellulase composition [128], microbial proteomes [129,130], a 1 -antitrypsin phenotypes in serum [131], proteins in human cerebrospinal fluid (CSF) [121], noncovalent protein complexes [132], isoenzymes of human alcohol dehydrogenase [133], FQlabeled green fluorescent protein [134], yeast cytosol proteins [135], HIV envelope glycoprotein [44], and protein heterogeneity caused by labeling with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate [29].…”

“…CIEF was employed to analyze erythropoietin glycoforms [120], BSA and its monoclonal antibody [211,212], heme proteins [85], Enbrel isoforms [125], proteins in single cell embryo of nematode Caenorhabditis elegans [213], cellobiohydrolase I in extracts from Trichoderma reesei [128], major erythrocyte membrane proteins [214], noncovalent protein-protein complexes [132], and isoenzymes of human alcohol dehydrogenase [133].…”

Recent Developments in Capillary Electrophoresis–Mass Spectrometry of Proteins and Peptides

“…A means to close the CE electrical circuit for analyte separation while simultaneously providing an electrical potential to the spray tip is necessary. The common interfaces can be divided into three categories: sheath-flow 16,[18][19][20]24,27,32,37,38,40,43,47,50,[53][54][55][56]60,66 and sheathless 15,17,25,26,[28][29][30]34,36,44,45,48,49,61 and liquid-junction interfaces. [92][93][94] In the sheath-flow design, a coaxial sheath liquid, usually a hydroorganic mixture, mixes with the capillary eluent at the outlet end of the capillary.…”

“…Each component is focused at the position in the pH gradient where it has a net zero charge. 4,110 The high-resolution capability of CIEF, frequently in conjunction with the high sensitivity, mass resolving power and mass measurement accuracy of an ESIFourier transform ion cyclotron resonance (FTICR) mass spectrometer, has been harnessed for characterizing complex protein mixtures, either alone 16,18,19,31 or in combination with other separation steps. 23,46,47,58 In-capillary electrophoretic concentration schemes can provide as much as three orders of magnitude improvement in the signal (e.g.…”

Review applications of capillary electrophoresis to the analysis of biotechnology-derived therapeutic proteins

“…Very subtle changes in a protein molecule can produce measurable changes in its isoelectric point as evident from the study of recombinant alcohol dehydrogenase isoforms by capillary isoelectric focusing (CIEF) [6]. Isoforms of the protein a-, b1-, b2-and b3 subunits were incubated together and were shown to form both homodimers and heterodimers.…”