To study incorporation of nonproteinogenic amino acids in bacterial proteome we performed a global, unbiased protein modification analysis of the E. coli K12 strain with defective editing mechanism of the leucyl tRNA synthetase (LeuRS), which in addition to leucine incorporates a nonproteinogenic amino acid norvaline. We measured widespread loss of methyl groups at leucine positions in the LeuRS mutant, indicative of norvaline incorporation. We performed a Super-SILAC experiment and established that under microaerobic conditions norvaline misincorporation reaches a maximum approximately 10 hours after entry into stationary phase, when up to 10% of all measured leucines are substituted with norvaline.