|Hajkova, Dagmar -|
|Hervey, William -|
|Deschamps, Jeffrey -|
|Kusterbeck, Anne -|
|Vora, Gary -|
Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 2, 2012
Publication Date: April 2, 2012
Citation: Hajkova, D., Hervey, W., Li, R.W., Deschamps, J., Kusterbeck, A., Vora, G. 2012. Method Development for Metaproteomic Analyses of Marine Biofilms. Analytical Chemistry. 84(9):4006–4013. Interpretive Summary: Despite decades of research that have focused on understanding the formation and prevention of biofilms, relatively little is known about the microbial consortia and biomolecular components that are responsible for biofilm formation on varying substrates and in differing seasonal and geographical environments. Biofilms play a critical role in microbial fermentation in the rumen. In this study, we developed methods and databases for high-throughput identification of peptides and proteins using a marine biofilm model. We demonstrated repeatable method-specific differences in the number of protein identifications and protein coverage and provided criteria to consider for qualitative or quantitative biofilm metaproteome experimental design. Our results will facilitate studies in bacterial biofilms associated with plant fiber and food particles in the rumen and the colon of humans and animals.
Technical Abstract: The large-scale identification and quantitation of proteins via nano-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) offers a unique opportunity to gain unprecedented insight into the microbial composition and biomolecular activity of true environmental samples. However, in order to realize this potential for marine biofilms, new methods of protein extraction must be developed as many compounds naturally present in biofilms are known to interfere with common proteomic manipulations and LC-MS/MS techniques. In this study, we used amino acid analyses (AAA) and LC-MS/MS to compare the efficacy of three protein extraction methods [using guanidine hydrochloride (GuHCl), sodium dodecyl sulfate (SDS), and phenol], two separation methods [1D-SDS-PAGE-LC and 2D-LC]and two digestion methods [in-solution and in-gel] for the metaproteomic analyses of an environmental marine biofilm. The AAA demonstrated that proteins constitute 1.24% of the biofilm wet weight and that the compared methods varied in their protein extraction efficiencies (0.85 – 15.15%). Subsequent LC-MS/MS analyses revealed that the GuHCl / in-solution digestion / 2D-LC method resulted in the greatest number of proteins identified by one or more peptides whereas the phenol / SDS-PAGE-1D-LC/ in-gel digestion method provided the greatest sequence coverage of identified proteins. As expected, metagenomic sequencing of the same biofilm sample enabled the creation of a searchable database that increased the number of protein identifications by 48.7% (=1 peptide) or 54.7% (=2 peptides) when compared to SwissProt database identifications. Taken together, our results provide methods and evidence-based recommendations to consider for qualitative or quantitative biofilm metaproteome experimental design.