Abstract
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.
Highlights
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
For pulse labeling applications under relevant physiological conditions it is important to know how E. coli grows on azhal and incorporates it into cellular proteins
The suitability of azhal as a pulse label was tested by measuring growth and viability of E. coli grown on a medium containing azhal as a substitute for methionine
Summary
Synthesis of L-Azhal—L-Azhal was synthesized from L-Boc-2,4diaminobutyric acid (L-Boc-DAB, Chem-Impex) by diazo transfer using triflic azide (TfN3) as described previously [30]. Peptide samples subjected to LC-tandem MS for protein identification with MASCOT contained both TCEP-induced reaction products from azhal-labeled peptides and unmodified peptides not derived from azhal-containing peptides by TCEP treatment To remove the latter species, we selected manually in MASCOT only those peptides that have the variable modifications homoserine lactone, diaminobutyrate, and homoserine and the unmodified C-terminal peptides that resulted from cleavage after a methionine residue. The resulting MASCOT data file of this search was imported into Quant [36] for quantitation using the iTRAQ reporter ions using only labeled peptides unique to each protein. For identification purposes both searches were exported as comma-separated value files. Ratios greater than 1 indicate relative over-representation of mapped proteins in the category of the newly synthesized proteins compared with the proteome, and ratios smaller than 1 represent relative under-representation
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