Because the increasing morbidity of pertussis in all age groups worldwide, the quality of pertussis vaccines has aroused a common concern. To improve the quality of pertussis vaccine in research and production, the effects of manufacture processes on post-translational modifications (PTMs) of bioactive proteins in pertussis vaccine were investigated by a liquid chromatography quadruple - time of flight mass spectrometer (LC-Q-TOF) method in this study. The main bioactive proteins in pertussis vaccine studied include pertussis toxin (PT), pertactin (PRN) and filamentous hemagglutinin (FHA). The main manufacture processes focused are fermentation techniques, purification techniques and storage conditions. The results show that FHA and PRN are rather stable against PTM as only deamidation (Asn) was detected, which is believed to be due to their larger sizes of the bioactive proteins. For PT, however, all the manufacture processes studied have shown significant effects on types and sites of PTMs. Modifications of oxidation and demethylation (Met) occurred in the PT proteins produced by B. pertussis strain Tohama and stored in suspension in saline solution. However, they were not observed in the PT samples produced from stain CS and stored in powders. Carbamylation (Arg) on multiple sites (in S3, S4 and S5) was observed in the PT produced from 5th generation strain CS of B. pertussis. The high abundance ratio of carbamylation modification was potentially a negative effect on the detoxification of PT, since unmodified Lys was the active site for detoxification. The results obtained in this study provide information for making protection strategies against PTMs in pertussis vaccine in manufacture and storage.