Analysis of the Amphibalanus amphitrite adhesive proteome using pressure cycling technology: Method development and the effect of extraction solvents

ABSTRACT: Background: Marine biofouling negatively impacts industries reliant upon submerged stationary surfaces. The acorn barnacle Amphibalanus amphitrite is a major biofouler that permanently attaches to a wide array of substrates by producing a proteinaceous adhesive at the surface interface. These interface proteins are difficult to solubilize, and the understanding of what proteins exist in the adhesive has only been expanded with the use of the strong polar solvent hexafluoroisopropanol in combination with standard gel-based sample processing methods for proteomics analysis. Although effective, existing sample processing methods are labor and time intensive, hindering progress in this field. Results: We have developed a more efficient sample processing method by exploiting pressure cycling technology, which aids in protein extraction and digestion for proteomics analysis, and explored the efficacy of multiple solvents in combination with pressure on protein identification. We found that barnacle adhesive proteins can be extracted and digested in the same tube using pressure cycling technology, minimizing sample loss, increasing throughput to 16 concurrently processed samples, and decreasing sample processing time to under 8 hours. Pressure cycling technology methods produced similar proteomes in comparison to previous methods. Two solvents which were ineffective at extracting proteins from the adhesive at ambient pressure (urea and methanol) produced more protein identifications under pressure than hexafluoroisopropanol, leading to the identification and our description of >40 novel proteins at the interface. Many A. amphitrite adhesive proteins have no sequence similarity to publically available proteins, highlighting the unique adherent processes evolved by barnacles. Conclusion: Using pressure cycling technology, we describe methods that produce robust and consistent protein identifications from barnacle adhesive. These methods can be used to examine changes in barnacle adhesive, enabling future research on the effects of environmental and surface substrate composition on barnacle cement, a critical step for both broadening the understanding of barnacle adhesion and the development of new coatings for submerged surfaces to combat hard biofoulants.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Amphibalanus Amphitrite (striped Barnacle) (balanus Amphitrite)

SUBMITTER: Janna Schultzhaus  

LAB HEAD: Janna Schultzhaus

PROVIDER: PXD012730 | Pride | 2019-07-04

REPOSITORIES: Pride