An optimized metaproteomics protocol for a holistic taxonomic and functional characterization of microbial communities from marine particles

Abstract

This study aimed to establish a robust and reliable metaproteomics protocol for an in-depth characterization of marine particle-associated (PA) bacteria. To this end, we compared six well-established protein extraction protocols together with different MSsample preparation techniques using particles sampled during a North Sea spring algae bloom in 2009. In the final optimized workflow, proteins are extracted using a combination of SDS-containing lysis buffer and cell disruption by bead-beating, separated by SDS-PAGE, in-gel digested and analysed by LC-MS/ MS, before MASCOT search against a metagenomebased database and data processing/visualization with the in-house-developed bioinformatics tools Prophane and Paver. As an application example, freeliving (FL) and particulate communities sampled in April 2009 were analysed, resulting in an as yet unprecedented number of 9354 and 5034 identified protein groups for FL and PA bacteria, respectively. Our data suggest that FL and PA communities appeared similar in their taxonomic distribution, with notable exceptions: eukaryotic proteins and proteins assigned to Flavobacteriia, Cyanobacteria, and some proteobacterial genera were found more abundant on particles, whilst overall proteins belonging to Proteobacteria were more dominant in the FL fraction. Furthermore, our data points to functional differences including proteins involved in polysaccharide degradation, sugar-and phosphorus uptake, adhesion, motility, and stress response.

Distinct Human Gut Microbial Taxonomic Signatures Uncovered With Different Sample Processing and Microbial Cell Disruption Methods for Metaproteomic Analysis

“…As for bead-beating, the suitable size of the beads to be used in gut microbiota studies is not well defined yet. Small beads (<0.5 mm) ( Wu et al, 2016 ; Zhang et al, 2018 ; Schultz et al, 2020 ) have been normally used in this type of studies, while bigger glass beads (>0.5 mm) are normally used for yeast cell disruption ( Pitarch et al, 2008 ). A mixture of different sized beads has been proven to better extract proteins from gram-positive bacteria and yeast ( Hayoun et al, 2019 ).…”

Identifying Robust Microbiota Signatures and Interpretable Rules to Distinguish Cancer Subtypes

“…Thiodiazotropha contributes to the identification of such cancer subtype with publication support. Terasakiispira is detected in the pathological urinary system, including the malignant transformed urinary system ( Schultz et al, 2020 ), and contributes to abnormal genomic alterations in human beings ( Angiuoli et al, 2008 ).…”

An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum

“…Thus, to further increase the amount of microbial proteins compared to human proteins, microbial cells were enriched, while human cells were depleted, prior to MS/MS analyses. Several approaches were tested using the sputum test samples to reduce sample complexity and enrich microbial cells: differential centrifugation (Tanca et al, 2014;Tanca et al, 2017), filtration (Xiong et al, 2015;Schultz et al, 2020), as well as density gradient centrifugation (Hevia et al, 2016). Each procedure was investigated for the enrichment success microscopically (Figure S2).…”