Total Protein Extraction for Metaproteomics Analysis of Methane Producing Biofilm: The Effects of Detergents

Abstract

Protein recovery is crucial for shotgun metaproteomics to study the in situ functionality of microbial populations from complex biofilms but still poorly addressed by far. To fill this knowledge gap, we systematically evaluated the sample preparation with extraction buffers comprising four detergents for the metaproteomics analysis of a terephthalate-degrading methanogenic biofilm using an on-line two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) system. Totally, 1018 non-repeated proteins were identified with the four treatments. On the whole, each treatment could recover the biofilm proteins with specific distributions of molecular weight, hydrophobicity, and isoelectric point. The extraction buffers containing zwitterionic and anionic detergents were found to harvest the proteins with better efficiency and quality, allowing identification up to 76.2% of total identified proteins with the LC-MS/MS analysis. According to the annotation with a relevant metagenomic database, we further observed different taxonomic profiles of bacterial and archaeal members and discriminable patterns of the functional expression among the extraction buffers used. Overall, the finding of the present study provides first insight to the effect of the detergents on the characteristics of extractable proteins from biofilm and the developed protocol combined with nano 2D-LC/MS/MS analysis can improve the metaproteomics studies on microbial functionality of biofilms in the wastewater treatment systems.

Tick and Host Derived Compounds Detected in the Cement Complex Substance

“…The analysis of peptides used to identify host and tick derived proteins (Data S7) evidenced some distinctive characteristics of the cementome tick-derived proteins (Table S3). These characteristics included a higher pI suggesting a more basic protein composition, lower aliphatic index associated with lower thermostability of globular proteins [52], and a lower GRAVY index suggesting the presence of less hydrophilic proteins [53]. The SEM combined with EDS was used for the analysis of chemical elements in R. microplus salivary glands and cement cones ( Figure 8A,B, Data S5 and S6).…”

Metaproteomic analysis using the Galaxy framework

“…At its inception [13], metaproteomics relied heavily on 2DE for protein separation [7,[14][15][16][17]. Subsequent methodological developments have focused on mixture fractionation [18][19][20] and protein extraction [5,[21][22][23][24][25] prior to shotgun LC MS/MS. Proteins identified from microbial communities have also been validated using a targeted proteomics approach [26].…”

“…These developments have led to detailed metaproteomic analysis in various artificial or natural ecosystems such as activated sludge [13,16,22], anaerobic degradation [7,33,34], biogas plants [18], wastewater systems [2,23]), water bodies contaminated with vinyl chloride [26], metals [8], or coal tarcontaminated groundwater [35]; aquatic lakes [36], ocean scale shifts in microbial nutrient utilization [37] to winter to spring succession in coastal Atlantic Ocean microbial plankton [38].…”

“…These developments have led to detailed metaproteomic analysis in various artificial or natural ecosystems such as activated sludge , anaerobic degradation , biogas plants , wastewater systems ), water bodies contaminated with vinyl chloride , metals , or coal tar‐contaminated groundwater ; aquatic lakes , ocean scale shifts in microbial nutrient utilization to winter to spring succession in coastal Atlantic Ocean microbial plankton . Additional metaproteomic analyses have studied the soil microbiome , with primary focus on optimization of protein extraction methods, and the study of rhizosphere or rice plant tissues .…”

Development of an Integrated Pipeline for Profiling Microbial Proteins from Mouse Fecal Samples by LC–MS/MS

“…10−12 In these studies, different sample preparation procedures were employed and differed in the application of bead−beating for protein extraction. Herein, by comparing the efficiency of protein identification with or without bead-beating, we confirmed that bead-beating is essential for sample preparation for metaproteomics analysis.…”