Direct measurement of islet amyloid polypeptide fibrillogenesis by mass spectrometry.
A novel method for monitoring fibrillogenesis is developed and applied to the amyloidogenic peptide, islet amyloid polypeptide (IAPP). The approach, based on electrospray ionization mass spectrometry, is complementary to existing assays of fibril formation as it monitors directly the population of precursor rather than product molecules. We are able to monitor fiber formation in two modes: a quenched mode in which fibril formation is halted by dilution into denaturant and a real time mode in which fibril formation is conducted within the capillary of the electrospray source. Central to the method is the observation that fibrillar IAPP does not compromise the ionization of monomeric IAPP. Furthermore, under mild ionization conditions, fibrillar IAPP does not dissociate and contribute to the monomeric signal. Critically, 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 sufficiently 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. Furthermore, a consistent level of monomer persists in both seeded and unseeded experiments after the fibril formation is complete. Given the inherent stability of fibrils, we expect this approach to be applicable to other amyloid systems.
Larson
J L
JL
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Ko
E
E
Miranker
A D
AD
D000682:Amyloid / Q000737:chemistry* / Q000592:standards; D000818:Animals; D001704:Biopolymers / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D058228:Islet Amyloid Polypeptide; D007515:Islets of Langerhans / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D013058:Mass Spectrometry / Q000592:standards; D051381:Rats; D012015:Reference Standards
D000682:Amyloid; D001704:Biopolymers; D058228:Islet Amyloid Polypeptide; D046911:Macromolecular Substances