Direct measurement of islet amyloid polypeptide fibrillogenesis by mass spectrometry

A novel method for monitoring fibrillogenesis is developed and applied to t he amyloidogenic peptide, islet amyloid polypeptide (IAPP). The approach, b ased on electrospray ionization mass spectrometry, is complementary to exis ting assays of fibril formation as it monitors directly the population of p recursor rather than product molecules. We are able to monitor fiber format ion in two modes: a quenched mode in which fibril formation is halted by di lution into denaturant and a real time mode in which fibril formation is co nducted within the capillary of the electrospray source. Central to the met hod is the observation that fibrillar IAPP does not compromise the ionizati on of monomeric IAPP. Furthermore, under mild ionization conditions, fibril lar IAPP does not dissociate and contribute to the monomeric signal. Critic ally, we introduce an internal standard, rat IAPP, for analysis on the mass spectrometer. This standard is sufficiently similar in sequence in that it ionizes identically to human IAPP. Furthermore, the sequence is sufficient ly different in that it does not form fibrils and is distinguishable on the basis of mass. Applied to IAPP fibrillogenesis, our technique reveals that precursor consumption in seeded reactions obeys first-order kinetics. Furt hermore, a consistent level of monomer persists in both seeded and unseeded experiments after the fibril formation is complete. Given the inherent sta bility of fibrils, we expect this approach to be applicable to other amyloi d systems.