Mass spectrometric analysis of eukaryotic proteosome
EMSL Project ID
4093
Abstract
Ubiquitin-dependent proteolysis is catalyzed by the 26S proteasome, a dynamic complex of 32 different proteins whose mode of assembly and mechanism of action is poorly understood. The 26S proteasome consists of a self-compartmentalized 20S protease core that is capped at one or both ends by the 19S regulatory particle, or cap. The 20S core particle is made up of two copies each of seven different alpha and beta subunits arranged into four stacked rings. The stoichiometry of these rings is highly conserved.The composition of eukaryotic 26S proteasomes has been analyzed in several studies. Most of the studies have relied on conventional chromatographic fractionation steps and two dimensional SDS-PAGE, followed by sequence analysis of individual polypeptides. Only a select few have chosen MS as an approach for proteasomal analysis. The most current and sensitive was the analysis of purified proteasomes by ion trap MS/MS (Verma, Rati., et al. Molecular biology of the cell , Vol 11. Oct, 2000). This study detected several new proteins associated with the proteasome.
Analysis of an affinity-purified yeast proteasome preparation by high resolution capillary LC-FTICR MS using the AMT tag approach would address two issues. First, the highly sensitive FTICR in combination with a high resolution LC tryptic peptide separation would provide highly confident identifications for all of the proteins in the affinity-purified yeast proteasome. Secondly, using newly developed software at PNNL, relative protein abundances could be determined providing a dataset that could subsequently be correlated with the reported stoichiometry of the proteasome subunits. This would be the first study that provides direct correlation of the MS relative protein abundances to reported subunit stoichiometries.
In addition to the determination of relative protein abundances it is anticipated that posttranslational modifications present in the proteasome proteins would also be detected using the SEQUEST algorithm applied to the ion trap MS/MS data generated in the initial step of the AMT tag method. However, further analysis of intact proteasomal proteins using CIEF-FTICR MS should reveal additional information on cotranslational and posttranslational modifications including loss of the initiating methionine, acetylation, methylation, phosphorylation and proteolytic maturation.
Affinity-purified proteasomes from several specific experimental conditions will be analyzed to further the understanding of the proteasome mechanism and its related substrate specificity.
Project Details
Project type
Exploratory Research
Start Date
2003-08-26
End Date
2006-08-27
Status
Closed
Released Data Link
Team
Principal Investigator