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

Doreen Schultz,Daniela Zühlke,Stephanie Markert,Claudia Hirschfeld,Jörg Bernhardt,Dirk Albrecht,Katharina Riedel,Thomas Ben Francis

doi:10.1111/1758-2229.12842

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 MS-sample 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 metagenome-based database and data processing/visualization with the in-house-developed bioinformatics tools Prophane and Paver. As an application example, free-living (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.

Highlights

A 20% of marine bacteria lives attached to algae or marine particles (Azam et al, 1983)
Whilst the metaproteomics analyses by Teeling et al (2012) and Kappelmann et al (2019) of FL bacterioplankton sampled during spring blooms from 2009 to 2012 off the German island Helgoland (5411003”N, 75400000E) resulted in the identification of several thousand protein groups, the PA microbial communities retained on 3 and 10 μm pore-sized filters were notably more difficult to analyse by the integrated metagenomic/metaproteomic approach employed at that time
We present a broadly applicable metaproteomics protocol for successful extraction of proteins from marine particles

Summary

Introduction

A 20% of marine bacteria lives attached to algae or marine particles (Azam et al, 1983) These marine particles consist of various kinds of organic matter, i.e. dead/ dying zoo- or phytoplankton, bacterioplankton, as well as inorganic small particles held together by a sugary matrix consisting of polysaccharide-composed transparent extracellular particles (TEPs) composed of polysaccharides, which are exuded mostly by phytoplankton and bacteria (Alldredge et al, 1993). Marine particles grow while sinking and contribute largely to the ‘biological pump’ by transporting carbon to deeper waters and sediments (Volkman and Tanoue, 2002) These aggregates may reach several centimetres in diameter. They are enzymatically well equipped to metabolize high-molecular weight substrates, providing nutrition to the attached community as well as leaving smaller carbon compounds to the surrounding water column community (Simon et al, 2002; Grossart, 2010)

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