Synthesis/degradation ratio mass spectrometry for measuring relative dynamic protein turnover.
One of the major unanswered questions in quantitative proteomics is that of dynamic protein turnover in the cell. Here we present a new approach to quantitative proteomics that measures the relative dynamic turnover of proteins in cellular systems. In this approach, termed synthesis/degradation ratio mass spectrometry, stable isotope labeling is employed to calculate a relative synthesis/degradation ratio that reflects the relative rate at which 13C is incorporated into individual proteins in the cell. This synthesis/degradation ratio calculation is based on a Poisson distribution model that is designed to support high-throughput analysis. Protein separation and analysis is accomplished by utilizing one-dimensional SDS-PAGE gel electrophoresis followed by cutting the gel into a series of bands for in-gel digestion. The resulting peptide mixtures are analyzed via solid-phase MALDI LC-MS and LC-MS/MS using a tandem time-of-flight mass spectrometer. A portion of the soluble protein fraction from an E. coli K-12 strain was analyzed with synthesis/degradation ratios varying from approximately 0.1 to 4.4 for a variety of different proteins. Unlike other quantitative techniques, synthesis/degradation ratio mass spectrometry requires only a single cell culture to obtain useful biological information about the processes occurring inside a cell. This technique is highly amenable to shotgun proteomics-based approaches and thus should allow relative turnover measurements for whole proteomes in the future.
Cargile
Benjamin J
BJ
Mass Spectrometry Research Group, Research Triangle Institute, 3040 Cornwallis Road, Research Triangle Park, North Carolina 27709-2194, USA.
Bundy
Jonathan L
JL
Grunden
Amy M
AM
Stephenson
James L
JL
D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence / Q000235:genetics; D013058:Mass Spectrometry / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001426:Bacterial Proteins