Structure specific amyloid precipitation in biofluids

Abstract

The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. Here we report a method to capture amyloid containing aggregates in human biofluids in an unbiased way, a process we name amyloid precipitation (AP). We use a structure-specific chemical dimer; a Y shaped, bioinspired small molecule with two capture groups for AP to increase affinity. Our capture molecule for amyloid precipitation (CAP-1), consists of a derivative of Pittsburgh compound B (dimer) to target the cross β-sheets of amyloids and a biotin moiety for surface immobilization. By coupling CAP-1 to magnetic beads, we demonstrate that we can target the amyloid structure of all protein aggregates present in human cerebrospinal fluid, isolate them for analysis and then characterise them using single-molecule fluorescence imaging and mass spectrometry. Amyloid precipitation enables unbiased determination of the molecular composition and structural features of the in vivo aggregates, formed in neurodegenerative diseases.