Proteome-wide alterations in Escherichia coli translation rates upon anaerobiosis.

Enzyme reprofiling in bacteria during adaptation from one environmental condition to another may be regulated by both transcription and translation. However, little is known about the contribution of translational regulation. Recently, we have developed a pulse labeling method using the methionine analog azidohomoalanine to determine the relative amounts of proteins synthesized by Escherichia coli in a brief time frame upon a change in environmental conditions. Here we present an extension of our analytical strategy, which entails measuring changes in total protein levels on the same time scale as new protein synthesis. This allows identification of stable and labile proteins and demonstrates that altered levels of most newly synthesized proteins are the result of a change in translation rate rather than degradation rate. With this extended strategy, average relative translation rates for 10 min immediately after a switch from aerobiosis to anaerobiosis were determined. The majority of proteins with increased synthesis rates upon an anaerobic switch are involved in glycolysis and pathways aimed at preventing glycolysis grinding to a halt by a cellular redox imbalance. Our method can be used to compare relative translation rates with relative mRNA levels at the same time. Discrepancies between these parameters may reveal genes whose expression is regulated by translation rather than by transcription. This may help unravel molecular mechanism underlying changes in translation rates, e.g. mediated by small regulatory RNAs.

Selective enrichment of azide-containing peptides from complex mixtures.

A general method is described to sequester peptides containing azides from complex peptide mixtures, aimed at facilitating mass spectrometric analysis to study different aspects of proteome dynamics. The enrichment method is based on covalent capture of azide-containing peptides by the azide-reactive cyclooctyne (ARCO) resin and is demonstrated for two different applications. Enrichment of peptides derived from cytochrome c treated with the azide-containing cross-linker bis(succinimidyl)-3-azidomethyl glutarate (BAMG) shows several cross-link containing peptides. Sequestration of peptides derived from an Escherichia coli proteome, pulse labeled with the bio-orthogonal amino acid azidohomoalanine as substitute for methionine, allows identification of numerous newly synthesized proteins. Furthermore, the method is found to be very specific, as after enrichment over 87% of all peptides contain (modified) azidohomoalanine.

Identification and quantitation of newly synthesized proteins in Escherichia coli by enrichment of azidohomoalanine-labeled peptides with diagonal chromatography.

A method is presented to identify and quantify several hundreds of newly synthesized proteins in Escherichia coli upon pulse labeling cells with the methionine analogue azidohomoalanine (azhal). For the first 30 min after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 min and digestion of total protein, azhal-labeled peptides are isolated by a retention time shift between two reversed phase chromatographic runs. The retention time shift is induced by a reaction selective for the azido group in labeled peptides using tris(2-carboxyethyl)phosphine. Selectively modified peptides are identified by reversed phase liquid chromatography and on-line tandem mass spectrometry. We identified 527 proteins representative of all major Gene Ontology categories. Comparing the relative amounts of 344 proteins synthesized in 15 min upon a switch of growth temperature from 37 to 44 degrees C showed that nearly 20% increased or decreased more than 2-fold. Among the most up-regulated proteins many were chaperones and proteases in accordance with the cells response to unfolded proteins due to heat stress. Comparison of our data with results from previous microarray experiments revealed the importance of regulation of gene expression at the level of transcription of the most elevated proteins under heat shock conditions and enabled identification of several candidate genes whose expression may predominantly be regulated at the level of translation. This work demonstrates for the first time the use of a bioorthogonal amino acid for proteome-wide detection of changes in the amounts of proteins synthesized during a brief period upon variations in cellular growth conditions. Comparison of such data with relative mRNA levels enables assessment of the separate contributions of transcription and translation to the regulation of gene expression.