Proteome dynamics in complex organisms: using stable isotopes to monitor individual protein turnover rates.
The complete definition of changes in a proteome requires information about dynamics and specifically the rate at which the individual proteins are turned over intracellularly. Whilst this can be achieved in single-cell culture using stable isotope precursors, it is more challenging to develop methods for intact animals. In this study, we show how dietary administration of stable isotope-labelled amino acids can obtain information on the relative rates of synthesis and degradation of individual proteins in a proteome. The pattern of stable isotope-labelling in tryptic peptides can be deconstructed to yield a highly reliable measure of the isotope abundance of the precursor pool, a parameter that is often difficult to acquire. We demonstrate this approach using chickens fed a semisynthetic diet containing [(2)H(8)]valine at a calculated relative isotope abundance (RIA) of 0.5. When the labelling pattern of gel-resolved muscle proteins was analyzed, the intracellular precursor isotope abundance was 0.35, consistent with dilution of the amino acid precursor pool with unlabelled amino acids derived from degradation of pre-existing proteins. However, the RIA was stable over an extended labelling window, and permitted calculation of the rates of synthesis and degradation of individual proteins isolated by gel electrophoresis. For the first time, it is feasible to contemplate the analysis of turnover of individual proteins in intact animals.
Doherty
Mary K
MK
Department of Veterinary Preclinical Sciences, University of Liverpool, Liverpool, UK.
Whitehead
Colin
C
McCormack
Heather
H
Gaskell
Simon J
SJ
Beynon
Robert J
RJ
D000818:Animals; D002645:Chickens; D003903:Deuterium / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D007553:Isotope Labeling; D007700:Kinetics; D009124:Muscle Proteins / Q000032:analysis* / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism; D014633:Valine / Q000008:administration & dosage / Q000378:metabolism
D009124:Muscle Proteins; D010455:Peptides; D020543:Proteome; D003903:Deuterium; D014357:Trypsin; D014633:Valine