Amino Acids Boost Protein Recovery in Microbial Communities
Scientists find optimal soil pretreatment methods
Studying the proteins expressed by soil-dwelling microbial communities helps to define their fundamental biogeochemical roles in carbon cycling, nitrogen cycling, phosphorus cycling, and climate regulation, as well as determine how they might assist with environmental cleanup.
Results: Scientists can now recover and identify twice as many proteins expressed by soil-dwelling microbes than they could previously thanks to a new method of soil pretreatment developed by a scientific team led by Pacific Northwest National Laboratory researchers.
The researchers tested a variety of methods and found that recovery and identification of proteins is significantly enhanced if, before lysis (dissolution), soil samples are treated with a cocktail of polar-positive amino acids, which bind to soil and block protein-binding sites. Upon lysis, a desorption buffer enhances the solubilization of proteins from the soil/solution mixture, while the amino acid pretreatment prevents protein from adsorbing onto the soil. The strategy for processing samples reveals new insight into the function of microbial communities in their native environments.
Why It Matters: The ability to identify twice as many proteins expressed by microbial communities in natural samples helps to define their fundamental biogeochemical roles in carbon cycling, nitrogen cycling, phosphorus cycling, and climate regulation. It also helps to determine how these communities might assist with environmental cleanup. For example, bacterial protein expression could be optimized to help remediate sites contaminated with toxic metals or radionuclides.
Methods: The scientists tested the methods on a model system consisting of Escherichia coli and uncontaminated soil samples from Rifle, Colorado; a variety of soil samples having different sand and silt compositions; and an indigenous soil sample contaminated with diesel oil from King George, Antarctica.
The method does not interfere with mass spectrometry analyses, and in the model system, the researchers showed that the polar-positive amino acid/desorption buffer pretreatment improved protein identification by nearly an order of magnitude compared to using no pretreatment at all.
Their results show that it is possible to significantly increase protein identifications by blocking binding sites on a variety of soil and sediment textures. Using an optimized desorption buffer further increases the number of identifications.
What's Next? The researchers are currently experimenting with different binders to further improve the effectiveness of blocking protein-binding sites on soil. The desorption buffer is also being optimized to solubilize proteins without co-extracting contaminating humic substances (organics in soil).
Support: This research was supported by the U.S. Department of Energy Office of Biological and Environmental Research (BER) Genomic Science Program. Portions of the work were performed at EMSL, the Environmental Molecular Sciences Laboratory, a BER-supported national scientific user facility located at PNNL.
User Facility: EMSL
Research Team: Carrie Nicora, Mary Lipton, Brian Anderson, Stephen Callister, Angela Norbeck, Sam Purvine, and Richard Smith, PNNL; Janet Jansson and Maude David, Lawrence Berkeley National Laboratory; Olivia Mason, Florida State University; and Diogo Jurelevicius, Universidade Federal do Rio de Janeiro.
Reference: Nicora CD, BJ Anderson, SJ Callister, AD Norbeck, SO Purvine, JK Jansson, OU Mason, MM David, D Jurelevicius, RD Smith, and MS Lipton. 2013. "Amino Acid Treatment Enhances Protein Recovery from Sediment and Soils for Metaproteomic Studies." Proteomics 13(18-19):1776-1785. DOI: 10.1002/pmic.201300003.