Structural Analysis of a Prokaryotic Ribosome Using a Novel Amidinating Cross-Linker and Mass Spectrometry

ABSTRACT: The structure of the Escherichia coli ribosome, a 2.5 MDa ribonucleoprotein complex containing more than 50 proteins, was probed using the novel amidinating cross-linker diethyl suberthioimidate (DEST) and mass spectrometry. Peptide cross-links derived from this complex structure were identified at high confidence (FDR 0.8%) from precursor mass measurements and collision-induced dissociation (CID) fragmentation spectra. The acquired cross-linking data were found to be in excellent agreement with the crystal structure of the E. coli ribosome. DEST cross-links are particularly amenable to strong cation exchange (SCX) chromatography, facilitating a large-scale analysis. SCX enrichment and fractionation were shown to increase the number of cross-link spectra matches in our analysis 10-fold. Evidence is presented that these techniques can be used to study complex interactomes.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Chao Ji  

LAB HEAD: James P. Reilly

PROVIDER: PXD003381 | Pride | 2015-12-23

REPOSITORIES: Pride