Mass spectrometric peptide mapping analysis and structural characterization of dihydrodiol dehydrogenase isoenzymes.
The direct molecular weight determination and structural analysis of polypeptides and peptide mixtures have become amenable by the recent development of fast atom bombardment (FABMS) and 252Cf-plasma desorption (PDMS) mass spectrometry. FABMS and PDMS peptide mapping, i.e., the direct analysis of peptide mixtures resulting from proteolytic digestion, have been developed as powerful methods for the structural characterization of epoxide-metabolizing isoenzymes. The major advantage of this approach is provided by the selectivity of the endoproteolytic cleavage, combined with the specific and accurate molecular weight determination of complex digest mixtures containing peptides up to several thousands daltons in size. Furthermore, the mass spectrometric peptide mapping analysis can be combined with a range of protein-chemical modification reactions and with sequential degradation such as by carboxypeptidases. Both FABMS and PDMS peptide mapping have already been successfully applied to the structural differentiation of glutathione transferase and epoxide hydrolase isoenzymes in cases where references sequence data for at least one isoenzyme form was available. In the application described here, for a series of dihydrodiol dehydrogenase (DDH) isoenzymes with hitherto undetermined primary structures, a direct correlation between the structural differentiation from peptide mapping data and differences in their substrate specificities could be demonstrated. The mass spectrometric peptide mapping analysis of isoenzymes proved to be an efficient basis for the elucidation of the structure of one major DDH isoenzyme form; partial sequence data for this protein are reported.
Gauss
C
C
Faculty of Chemistry, University of Konstanz, Federal Republic of Germany.
Klein
J
J
Post
K
K
Suckau
D
D
Schneider
K
K
Thomas
H
H
Oesch
F
F
Przybylski
M
M
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010449:Peptide Mapping; D011817:Rabbits
D000596:Amino Acids; D007527:Isoenzymes; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C023125:dihydrodiol dehydrogenases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C116788:cis-1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase