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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.
