Soil microbial communities are some of the most daunting biochemical systems to study due to high taxonomic diversity and high metabolic diversity. Since most microbes are not culturable, one of the unique challenges in trying to decipher the physiology of these complex communities is determining which microbes are involved in specific biochemical processes. One of the main biochemical functions of soils is decomposition, which is driven by extracellular enzymes (exoenzymes). Our goal was to develop an effective method to separate exoenzymes from the abundant humic acids found in soils and then identify the exoenzymes using metaproteomics. Using soil cores obtained from Barrow, Alaska, we tested several methods to extract exoenzymes, remove as many humic acids as possible, digest the complex proteome into peptides and analyze by 2d-LC-MS/MS. Our focus is to improve method sensitivity, robustness, reproducibility, and dynamic range. We tested several key steps in the protocol including: bead beating/Mo-Bio Kit, Boiling/Non-Boiling, SDS/ SDC, FASP/Solution Digests and MW filters to remove humic acids. Samples were analyzed at the intact protein stage via 1D-SDS-PAGE and then further processed via Trypsin based proteome digestion into complex peptide solutions that were analyzed via 2d-LC-MS/MS (QExactive Plus).
We found the best protein recovery from our samples using SDS, boiling, FASP and a VivaSpin molecular weight filter to remove humic acids. Inspection of the raw chromatograms and the full scan spectra show large amounts of +1 ions, which represent small molecules that appear to not be peptides. However, there were a few +2 ions in the full scan spectra. Although not as abundant as the +1 ions, they likely represent the peptides we are targeting. The SDS-PAGE results corresponded to the FASP and solution digest results, showing proteins at lower molecular weight. Using this approach, we have recovered one test enzyme added to our soil samples and plan to continue to refining this protocol. For now, we are aware that sonification will not be incorporated in the protocol due denaturing of proteins. Our final goal is a robust integrated MS-based Metaproteomics methodology that is relatively straightforward, rapid and can process large numbers of samples providing exoenzymes identification and quantification.