This dataset contains spectral information of protein N-terminal peptides isolated from Listeria monocytogenes EGD-e, a bacterial model organism and human pathogen. When mapped onto the Listeria genome these peptides indicate the exact location of translation initiation sites (TIS). The large majority of the identified TIS corresponded to start sites of predicted open reading frames (ORFs), however, a significant fraction of the identified TIS indicated deviations from the current genome annotation. The latter include primarily TIS inside the sequence of predicted ORFs or TIS that delineate the start position of novel ORFs.