Determination of fractional synthesis rates of mouse hepatic proteins via metabolic 13C-labeling, MALDI-TOF MS and analysis of relative isotopologue abundances using average masses.
Proteins of a liver extract taken from a metabolically (13)C-labeled mouse were separated by 2D-PAGE and identified after tryptic digestion by MALDI-TOF MS peptide mass fingerprinting. (13)C-Labeling of proteins was achieved by an infusion of U-(13)C-glucose, which is metabolized to labeled nonessential amino acids. The labeling was analyzed using the relative isotopologue abundances of the measured isotope pattern of tryptic peptides and quantified by their increase in the average molecular mass (DeltaAVM). Fractional synthesis rates (FSR) of proteins were determined from corresponding peptides using measured DeltaAVM values as well as DeltaAVM values deduced from tRNA-precursor amino acid labeling, which in turn was derived from proteins showing high (13)C enrichments. The 8-h FSR values of 43 proteins were determined to range from 0 +/- 0.6 to 95 +/- 1%/8 h, with typical errors given as SEM values, which depend on the number of peptides of a specific protein usable for calculation. The method demonstrates that FSR values as an indicator for protein turnover in the liver proteome can be estimated within narrow error margins, providing baseline values from which treatment-dependent deviations could be detected with high statistical certainty.
Vogt
Josef A
JA
Universitätsklinikum für Anästhesiologie, Universität Ulm, Sektion APV, Parkstrasse 11, 89075 Ulm, Germany.
Hunzinger
Christian
C
Schroer
Klaus
K
Hölzer
Kerstin
K
Bauer
Anke
A
Schrattenholz
André
A
Cahill
Michael A
MA
Schillo
Simone
S
Schwall
Gerhard
G
Stegmann
Werner
W
Albuszies
Gerd
G
D000418:Albumins / Q000378:metabolism; D000818:Animals; D002247:Carbon Isotopes; D005591:Chemical Fractionation; D015180:Electrophoresis, Gel, Two-Dimensional; D005947:Glucose / Q000493:pharmacokinetics; D007553:Isotope Labeling / Q000379:methods; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010449:Peptide Mapping; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000032:analysis / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000418:Albumins; D002247:Carbon Isotopes; D011498:Protein Precursors; D011506:Proteins; D005947:Glucose