Direct identification of amyloids by label-free quantitative LC-MS

Direct identification of amyloids by label-free quantitative LC-MS
H. N. Danielsen, S. H. Hansen, F.-A. Herbst, P. H. Nielsen, M. S. Dueholm
Amyloids are highly ordered fibrillar protein polymers used by organisms from all domains of life due to their exceptional properties. We have previously shown that amyloids are widespread among microorganisms in biofilms from various habitats. They are therefore believed to play important roles in biofilm ecology. Despite their common appearance in biofilms, few amyloids have been characterized from biofilm-associated bacteria. However, the few amyloids that have been studied so far have already provided an astonishing demonstration of how the amyloids can be exploited with roles ranging structural components of biofilms, cell envelopes and spore coats to cytotoxins and as reservoirs for quorum-sensing signaling molecules.
The identification of novel functional amyloids is key to understand the many roles of amyloid in biofilms. Isolation of amyloids is unfortunately not a straightforward task. The insolubility and extreme stability of most functional amyloids exclude them from traditional protein analyses. Many functional amyloids are also highly adhesive and therefore bind to pipette tips and other consumables. Pure cultures, large sample volumes and high productivity of amyloids are therefore required for successful purification. We here present a quantitative proteomics technique that allow direct identification of functional amyloid candidates in complex samples based on their structural stability in the presence of increasing concentrations of formic acid.