Evaluation of protein extraction methods to improve meta-proteomics analysis of treated wastewater biofilms

Metaproteomics can be used to study functionally active biofilm-based bacterial populations in reclaimed water distribution systems, which in turn result in bacterial regrowth that impacts the water quality. However, existing protein extraction methods have differences in their protein recovery and have not been evaluated for their efficacies in reclaimed water biofilm samples. In this study, we first evaluated six different protein extraction methods with diverse chemical and physical properties on a mixture of bacterial cell culture. Based on a weighting scores-based evaluation, the extraction protocols in order of decreasing performance are listed as B-PER > RIPA > PreOmics > SDS > AllPrep > Urea. The highest four optimal methods on cell culture were further tested against treated wastewater non-chlorinated and chlorinated effluent biofilms. In terms of protein yield, our findings showed that RIPA performed the best; however, the highest number of proteins were extracted from SDS and PreOmics. Furthermore, SDS and PreOmics worked best to rupture gram-positive and gram-negative bacterial cell walls. Considering the five evaluation factors, PreOmics obtained highest weighted score, indicating its potential effectiveness in extracting proteins from biofilms. This study provides the first insight into evaluating protein extraction methods to facilitate metaproteomics for complex reclaimed water matrices.