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 azido homoalanine (azhal). For the first 30 minutes after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 minutes 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-carboxy-ethyl)-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 minutes upon a switch of growth temperature from 37 ºC to 44 ºC showed that nearly 20% in- or decreased more than two-fold. Amongst 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 micro-array 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.