Direct identification of functional amyloid proteins in cell lysate by label-free quantitative mass spectrometry

Functional amyloids are important structural and functional components in many biofilm, yet our knowledge of these fascinating polymers is limited to a few examples for which the amyloid have been purified and characterized by mass spectrometry. Isolation of the functional amyloid from other cell components represents the major bottleneck in the identification of novel functional amyloids. Here we present a label-free quantitative mass spectrometry based method that allows identification of amyloid proteins directly in cell lysates treated with increasing concentrations of formic acid (0-100%). Many functional amyloids are only depolymerized in the presence of concentrated formic acid and provide a characteristic sigmoidal signature when protein abundance is plotted against the formic acid concentration. An automated data processing pipeline was developed to provide a shortlist of amyloid protein candidates based on the amyloid specific abundance signature. The method was evaluated using the E. coli curli and the Pseudomonas Fap system. The major amyloid subunit was confidently identified for both systems and the minor subunit was also identified for the curli system. In addition, very few false positive candidates were identified and these could easily be discarded bases on bioinformatics analysis. Finally, we provide evidence for the application of the method for complex samples with low diversity. This provides an opportunity to identify amyloid in e.g. clinical biofilms.