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

Abstract

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.

Immunoproteomic Identification and Serological Responses to Novel Chlamydia pneumoniae Antigens That Are Associated with Persistent C. pneumoniae Infections

“…With the resulting monoisotopic peptide masses, database searches were performed using MASCOT search engine (www.matrixscience.com) as well as the ProFound search engine (http://prowl.rockefeller.edu/) with restriction to mass tolerances of maximal 10 ppm. Some protein samples were analyzed by ProteoSys by MALDI-TOF peptide mass fingerprinting, as published previously (42).…”

Proteome Dynamics: Revisiting Turnover with a Global Perspective

“…There are many caveats that should be attached to such calculations, but they serve to illustrate that in rapidly growing cells the degradative processes might be largely idling and handling the degradation of only a few key proteins plus any misfolded/damaged proteins. Studies performed with non-dividing cells (26) or adult animals (22,25,26,31,43,44,(62)(63)(64)(65)(66)(67) might give a more realistic picture of steady-state protein turnover, reflecting the process in cells that are non-dividing and which must adjust the intracellular protein abundance via manipulation of intracellular turnover. Although experimentally more challenging, the recent largescale studies obtained with complex systems (22,25,26,31,43,(65)(66)(67) are beginning to give insights into the maintenance of individual protein abundance in tissues, in organelles, and in species-a proteome-scale test of those observations initially made with total protein.…”

Precision Quantitative Proteomics with Fourier-Transform Mass Spectrometry

“…Advancement of high resolution proteomic technologies has provided the possibility to study protein turnover for multiple proteins simultaneously in complex cellular protein extracts (Beynon, 2005;Cargile et al, 2004;Pratt et al, 2002;Rao et al, 2008a;Rao et al, 2008b;Vogt et al, 2005). We showed that a combination of protein abundance and turnover data provides a highly interesting insight into the dynamic process of and interconnection among protein synthesis, degradation, and secretion (Rao et al, 2008a).…”