Motivation and aims

Understanding microbial community (MC) dynamics is key to advancing environmental microbiology, human health, and biotechnology. Metaproteomics analyzes all proteins in an MC, providing taxonomic and functional insights. Microbial activity and adaptation involve the synthesis of new proteins (nP), but differentiating nP from bulk proteins is challenging. Applying bioorthogonal non-canonical amino acid tagging (BONCAT) with click chemistry has demonstrated efficacy in enriching nP in pure cultures. However, transferring this technique to MC has been proven challenging due to inefficient BONCAT processes, resulting in low nP yield and high contamination from non-labeled bulk proteins. To address this issue, a new workflow to enrich nP from complex MC was developed. Initially tested with E. coli (labeled) and yeast (unlabeled control), the workflow was applied to an anaerobic MC from a laboratory biogas reactor.

Methods

After shifting from glucose to lactose, E. coli proteins were labeled with 4-Azido-L-homoalanine (AHA) and mixed with unlabeled yeast. AHA-labeled proteins were biotinylated via click chemistry, enriched using magnetic streptavidin beads, digested, and analyzed using LC-MS/MS. Protein identification was performed with MASCOT and MetaProteomAnalyzer. The workflow was then applied to an anaerobic MC shifted from glucose to ethanol, labeling the nP with AHA.

Results and discussion

Using breakable biotin linkers, amino acid bead blocking, and optimized washing resulted in specific enrichment of nP from E. coli (555 E. coli, 21 yeast in enriched sample versus 885 E. coli, 590 yeast in non-enriched sample). In the unlabeled control, only 7 proteins were detected. Applying the optimized workflow to the anaerobic MC identified 441 metaproteins, compared to 12 in the non-labeled control. Identifying a bifunctional alcohol dehydrogenase and syntrophic interactions between an ethanol-utilizing bacterium and two methanogens (hydrogenotrophic and acetoclastic) demonstrates the potential of metaproteomics targeting nP to trace microbial activity in complex MC.