Determination of Fractional Synthesis Rates of Mouse Hepatic Proteins via Metabolic ~(13)C-Labeling, MALDI-TOF MS and Analysis of Relative Isotopologue Abundances Using Average Masses

摘要

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 (ΔAVM). Fractional synthesis rates (FSR) of proteins were determined from corresponding peptides using measured ΔAVM values as well as ΔAVM 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.