title: Synthesis/degradation ratio mass spectrometry for measuring relative dynamic protein turnover.
abstract: 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.
authors.affiliation: Mass Spectrometry Research Group, Research Triangle Institute, 3040 Cornwallis Road, Research Triangle Park, North Carolina 27709-2194, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Cargile
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Bundy
authors.forename: Amy M
authors.initials: AM
authors.lastname: Grunden
authors.forename: James L
authors.initials: JL
authors.lastname: Stephenson
mesh_terms: 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*
chemical_list: D001426:Bacterial Proteins