Correct pairing of amino acids and tRNA is prerequisite for correct translation of genetic information during protein biosynthesis. Here, we present effects of proteome-wide isoleucine mistranslation in Escherichia coli induced by editing-defective isoleucyl-tRNA synthetase (IleRS). Two types of mistranslation were investigated: substitution of isoleucine (Ile) with either canonical (valine) or noncanonical (norvaline) amino acid. The effects of Ile to Val and Ile to Nva mistranslation were explored on proteome level. High level of Ile substitutions were achieved in editing-deficient strain, leading to excessive protein aggregation. Protein aggregates were analized by LC-MS/MS. Also, interaction of mistranslated proteins with DnaK was explored. The DnaK clients were isolated by pull-down, using endogenously expressed His-tagged DnaK.