Metabolically stable isotope labeling prior to electrophoretic protein separation reveals differences in fractional synthesis rates between mitochondrial aldehyde dehydrogenase isoforms.
Living mice were subjected to whole body labeling by intravenous infusion of [(13)C]glucose as the sole carbon source. After 10 h infusion the mice were sacrificed, and liver proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Five spots were found to contain mitochondrial aldehyde dehydrogenase (ALDH2) by matrix assisted time of flight mass spectrometry protein identification. By measuring the isotopologue mass distributions of peptide ions, and modeling the (13)C content of the precursor amino acid pool, the fractional synthesis rate of ALDH2 molecules synthesized during the labeling period was determined. One of the five spots was observed to have a five-fold higher fraction of (13)C-containing newly synthesized ALDH2 than the spot with the highest ALDH2 content, and contained more than 60% of newly synthesized ALDH2 although it accounted for less than 20% of the total ALDH2 detected. The total range in the fraction of (13)C-containing proteins between different ALDH2 spots approached 50-fold. The ability to quantitatively characterize different protein isoforms of biological origin for ALDH2 and other proteins from living animals provides new avenues for the exploration of protein function.
Cahill
Michael A
MA
ProteoSys AG, Carl-Zeiss-Strasse 51, D-55129 Mainz, Germany.
Vogt
Josef A
JA
Servos
Jörg
J
Wozny
Wojciech
W
Schwall
Gerhard P
GP
Groebe
Karlfried
K
Schrattenholz
André
A
Stegmann
Werner
W
D000444:Aldehyde Dehydrogenase / Q000096:biosynthesis* / Q000302:isolation & purification; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D007527:Isoenzymes / Q000096:biosynthesis* / Q000302:isolation & purification; D007553:Isotope Labeling; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007527:Isoenzymes; D000444:Aldehyde Dehydrogenase