We used nanoLC–MS/MS in a shotgun proteomics approach in order to identify the proteins expressed by P. nicotinovorans in the presence of nicotine at 3 different time intervals: 7, 10 and 24 hours post inoculation. A total of 915 proteins grouped 584 non-redundant clusters in were identified with an FDR of 0.3% when P. nicotinovorans was grown in on citrate medium alone and on citrate supplemented 0.05% nicotine. The differences in protein expression pattens at different time intervals allows us have a more dynamic picture on how the nicotine catabolic pathway is activated and to related it to the appearance of the known metabolic intermediates.