title: Ion mobility spectrometry-mass spectrometry defines the oligomeric intermediates in amylin amyloid formation and the mode of action of inhibitors.
abstract: The molecular mechanisms by which different proteins assemble into highly ordered fibrillar deposits and cause disease remain topics of debate. Human amylin (also known as islet amyloid polypeptide/hIAPP) is found in vivo as amyloid deposits in the pancreatic islets of sufferers of type II diabetes mellitus, and its self-aggregation is thought to be a pathogenic factor in disease and to contribute to the failure of islet transplants. Here, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to monitor oligomer formation from IAPP. The detection, identification and characterization of oligomers from both human and rat amylin (rIAPP) are described. Oligomers up to and including hexamers have been detected for both peptides. From ESI-IMS-MS derived collision cross sections (CCS), these species are shown to be elongated in conformation. Collision-induced dissociation (CID-MS/MS) revealed differences in the gas-phase stability of the oligomers formed from hIAPP and rIAPP, which may contribute to their differences in amyloid propensity. Using ESI-IMS-MS, the mode of inhibition of amyloid formation from hIAPP using small molecules or co-incubation with rIAPP was also investigated. We show that the polyphenolic compounds epigallocatechin gallate (EGCG) and silibinin bind to specific conformers within a dynamic ensemble of hIAPP monomers, altering the progress of oligomerization and fibril assembly. Hetero-oligomer formation also occurs with rIAPP but leads only to inefficient inhibition. The results indicate that although different small molecules can be effective inhibitors of hIAPP self-assembly, their modes of action are distinct and can be distinguished using ESI-IMS-MS.
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds , Leeds LS2 9JT, U.K.
authors.forename: Lydia M
authors.initials: LM
authors.lastname: Young
authors.forename: Ping
authors.initials: P
authors.lastname: Cao
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Raleigh
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
mesh_terms: D000818:Animals; D006801:Humans; D058228:Islet Amyloid Polypeptide / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis; D008024:Ligands; D051381:Rats; D000077385:Silybin; D012838:Silymarin / Q000737:chemistry / Q000494:pharmacology*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship
chemical_list: D058228:Islet Amyloid Polypeptide; D008024:Ligands; D012838:Silymarin; D054852:Small Molecule Libraries; D000077385:Silybin