Updated publication reference for PubMed record(s): 28887476, 26017780. A proteome-wide analysis was performed in Escherichia coli to identify the impact on protein N-termini of the antibiotic actinonin specifically inhibiting peptide deformylase (PDF). A new strategy and tool suite (SILProNaQ) was employed to provide large scale N-terminus acetylation yield quantitation. In control conditions, more than 1000 N-termini could be identified with 56 % Met removal, and additional modifications involving partial or complete N-acetylation (10%) and formyl retention (5%). Among the proteins undergoing these N-terminal modifications, some translocated membrane proteins were highlighted. The early time-course impact of actinonin was followed after the addition of bacteriostatic concentrations of the drug immediately slowing down the growth rate. Under these conditions, 25% of all proteins remain formylated after 10 min, a value reaching more than 60% of all characterized proteins after 40 min of treatment. The N-formylation rate on individual proteins increased with the same trend. Upon PDF inhibition, we finally show that two major categories of proteins retain their formyl group: a large number of inner membrane proteins and proteins involved in protein synthesis including many factors assisting the nascent chains in co-translational events.