Abstract
The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin.