2 research outputs found
Method Development for Metaproteomic Analyses of Marine Biofilms
The large-scale identification and quantitation of proteins
via
nanoliquid 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
sample preparation methods [6 M guanidine hydrochloride (GuHCl) protein
extraction + in-solution digestion + 2D LC; sodium dodecyl sulfate
(SDS) protein extraction + 1D gel LC; phenol protein extraction +
1D gel LC] 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 method resulted in the greatest number
of proteins identified by one or more peptides whereas the phenol
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
Method Development for Metaproteomic Analyses of Marine Biofilms
The large-scale identification and quantitation of proteins
via
nanoliquid 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
sample preparation methods [6 M guanidine hydrochloride (GuHCl) protein
extraction + in-solution digestion + 2D LC; sodium dodecyl sulfate
(SDS) protein extraction + 1D gel LC; phenol protein extraction +
1D gel LC] 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 method resulted in the greatest number
of proteins identified by one or more peptides whereas the phenol
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