The aim of our study is to understand the controllers of plant protein turnover in soil and in turn the factors dictating organic nitrogen recycling and usage in terrestrial ecosystems. Most temperate and boreal forest ecosystems are nitrogen limited and the decomposition of dead plant material is the largest input of organic nitrogen into the system. However, the processes that break down the macromolecular organic nitrogen compounds from decomposition into usable forms for plants and microbes has not received a lot of attention. The largest fraction of soil organic nitrogen is in the form of proteins, and therefore the controlling factors that regulate how these proteins can be broken down, from different microbial groups and different interactions with soil minerals, influences the bioavailability of nitrogen to the ecosystem and therefore its productivity. In this study we have developed a step-wise protocol for soil protein extraction in order to determine the fate of 15N-labeled ‘substrate’ proteins with distinct physiochemical characteristics in soils across a gradient of resource availability, mineralogy, and microbial ecology, with emphasis on the relative importance of protein-mineral and protein-organic matter interactions.
Four soil types across Oregon were used in a multi-step fractionation method for extracting proteins from soil. The soils ranged in pH (4-7), clay content (5-35%), organic carbon (7-87 g C kg-1 soil), organic nitrogen (0.6-5.7 g N kg-1 soil), and mean annual precipitation (400-2500 mm) to give a wide distribution of soil mineralogy. This step-wise approach was developed to achieve quantitative extraction of proteins from the soil matrix by targeting and disrupting different bonding interactions (e.g., physical occlusion, hydrogen bonding, electrostatic interactions, hydrophobic bonds) between polypeptide chains and soil matrix components. Initial experiments demonstrate that the soluble protein fraction accounts for 20-40% of proteins per extraction, the ionic bound fraction accounts for <5-20%, and hydrophobic bound fraction between 50-60%. The soils with the lowest organic matter and clay material had the lowest protein extraction efficiency, extracting about 20 ug protein/g soil versus 60-70 ug protein/g soil in contrasting soils. Comparatively, the Mo-Bio Protein Isolation Kit had a much lower protein extraction yield, between 5-25 ug protein/g soil. However, the step-wise method only extracted roughly 0.5-20% of proteinaceous organic nitrogen. Therefore, a targeted protein recovery experiment will be executed using 15N-labeled Douglas fir proteins to assess extraction efficiency across the four soil types with mass spectrometry analysis in order to optimize our protein extraction method.