Updated project metadata. In this study the impact of protein fractionation techniques prior to LC/MS analysis was investigated on activated sludge samples derived at winter and summer condition from a full-scale wastewater treatment plant (WWTP). For reduction of the sample complexity, different fractionation techniques including RP-LC (1D-approach), SDS-PAGE and RP-LC (2D-approach) as well as RP-LC, SDS-PAGE and liquid IEF (3D-approach) were carried out before subsequent ITMS analysis. The derived spectra were identified by MASCOT search using a combination of the public UniProtKB/Swiss-Prot protein database and metagenome data from a WWTP (data are available via ProteomeXchange with identifier PXD001547). The results showed a significant increase of identified spectra, enabled by applying IEF and SDS-PAGE to the proteomic workflow. Based on meta-proteins, a core metaproteome and the corresponding taxonomic profile of the wastewater activated sludge was revealed and functional aspects using the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway library were analyzed. Hereby, the KEGG Orthology identifiers (KO numbers) of protein hits were plotted into pathway maps of the central carbon (map01200) and nitrogen metabolism (map00910). Using the 3D-approach, most proteins involved in glycolysis and citrate cycle and nearly all proteins of the nitrogen removal were identified, qualifying this approach as the most promising for future studies.