Development of a Quartz Sand Protocol for Exoproteome Exploration from Anammox Consortia

A critical prerequisite for unbiased anaerobic ammonium oxidation (anammox) exoproteome exploration is the optimal extraction and treatment of extracellular polymeric substances (EPS). Herein, by leveraging the advantages of physical shear and potential electrostatic repulsion mechanisms, a non-destructive quartz sand protocol for in situ extraction of extracellular proteins (PN) for anammox consortia was developed and applied. According to an L-16(4(5)) orthogonal design, the optimal procedure, which contributes the maximum yield of EPS with minimal cell rupture, was identified as follows: 1.0 mm particle size, 100 g/g-VSS quartz sand ratio, 400 rpm, and 2 h. While achieving approximately the same yield of EPS, hierarchical clustering and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the quartz sand extraction was highly similar to the conventional cation exchange resin (CER) treatment in protein secondary structure properties and protein band distribution patterns, compared to other methods. However, the number of identified proteins and hydrophobic protein proportions were significantly increased by 5.3-fold and 2.6-fold after quartz sand treatment (122, 11.5%), respectively, compared to that of the CER method (23, 4.4%), while low cell lysis (52.1% extracellular component) was obtained based on subcellular localization data. Strong correlations between hydrophobicity of method-specific proteins and beta-sheet proportion (r(2) = 0.97, p = 0.0024), as well as architecture tightness (r(2) = 0.94, p = 0.0069) suggest that once the EPS protein structure becomes loose, exposed inner beta-sheet residues are prone to express hydrophobic properties. Addition of surfactants exhibited an undesirable solubilization effect on the protein secondary structure, gel separation, and spatial proteome, irrespective of PN yields. Overall, this work demonstrates the feasibility and effectiveness of extracting anammox EPS using an in situ quartz method that can satisfy yield-lysis trade-offs, retain the natural EPS traits, and allow the collection of more extracellular proteomic information.