Complementary protein extraction methods increase the identification of the Park Grass Experiment metaproteome

Although the Park Grass Experiment is an important international reference soil for temperate grasslands, it still lacks the direct extraction of its metaproteome. The identification of these proteins can be crucial to our understanding of soil ecology and major biogeochemical processes. However, the extraction of protein from soil is a technically fraught process due to difficulties with co-extraction of humic material and lack of compatible databases to identify proteins. To address these issues, we combined two protein extraction techniques on Park Grass experiment soil, one based on humic acid removal, namely a modified freeze-dry, heat/thaw/phenol/chloroform (HTPC) method and another which co-extracts humic material, namely an established surfactant method. A broad range of proteins were identified by matching the mass spectra of extracted soil proteins against a tailored Park Grass proteome database. These were mainly in the categories of “protein metabolism”, “membrane transport”, “carbohydrate metabolism”, “respiration” “ribosomal and nitrogen cycle” proteins, enabling reconstitution of specific processes in grassland soil. Protein annotation using NCBI and EBI databases inferred that the Park Grass soil is dominated by Proteobacteria, Actinobacteria, Acidobacteria and Firmicutes at phylum level and Bradyrhizobium, Rhizobium, Acidobacteria, Streptomyces and Pseudolabrys at genus level. Further functional enrichment analysis enabled us to connect protein identities to regulatory and signalling networks of key biogeochemical cycles, notably the nitrogen cycle. The newly identified Park Grass metaproteome thus provides a baseline on which future targeted studies of important soil processes and their control can be built.