The performance of a lab-scale wastewater treatment plant during the start-up phase was investigated. A period of varying pH resulted in the loss of ammonium removal efficiency together with a decrease in the specific autotrophic oxygen uptake rate (OUR). From the OUR, it was inferred that the ammonium oxidizing bacteria (AOB) were inhibited by the fluctuation in the pH values. However, OUR alone could not provide the information as to how the AOB were affected at the molecular level. To gain a better insight, shotgun proteomic method was used in this work to quantify the total proteins in the system. Label-free quantification (LFQ) showed that during the time of poor ammonium removal, the marker enzyme hydroxylamine oxidase found in Nitrosomonas sp. was at the lowest LFQ intensity. Based on these results, proteomics has the potential to be used as a monitoring tool. Nevertheless, there are still some restrictions when measuring activated sludge using proteomic method such as the availability of a suitable proteomic database. In this paper, we describe our experience of using publicly available database for identification of activated sludge proteins.