Early stages of insulin fibrillogenesis examined with ion mobility mass spectrometry and molecular modelling

Abstract

A prevalent type of protein misfolding causes the formation of β-sheet-rich structures known as amyloid fibrils. Research into the mechanisms of fibril formation has implications for both disease prevention and nanoscale templating technologies. This investigation into the aggregation of insulin utilises ion mobility mass spectrometry coupled with molecular modelling to identify and characterise oligomers formed during the 'lag' phase that precedes fibril growth. High resolution mass spectrometry and collision induced dissociation is used to unequivocally assign species as m/z coincident multimers or confomers, providing a robust analytical approach that supports the use of molecular dynamics to atomistically resolve the observed oligomers. We show that insulin oligomerises to form species In where 2 ≤ n ≤ 12 and within this set of oligomers we delineate over 60 distinct conformations, the most dominant of which are compact species. Modelling trained with experimental data suggests that the dominant compact dimers are enriched in β-sheet secondary structure and dominated by hydrophobic interactions, and provides a linear relationship between Rg and collision cross section. This approach provides detailed insight to the early stages of assembly of this much studied amyloidogenic protein, and can be used to inform models of nucleation and growth.

Native mass spectrometry—A valuable tool in structural biology

“…Prominent examples are epitope mapping utilising IM separation to identify antigen-epitope complexes [174][175][176] or fibril and amyloid formation. [177][178][179][180] Recent improvements in sample preparation (see Section 2) even allow the native IM-MS analysis of proteins from cell lysates. 21 The possibility of high-resolution native MS revolutionised the field of native MS and extended its applications enormously.…”

“…It ranges from building structural models to functional studies. Prominent examples are epitope mapping utilising IM separation to identify antigen‐epitope complexes 174–176 or fibril and amyloid formation 177–180 . Recent improvements in sample preparation (see Section 2) even allow the native IM‐MS analysis of proteins from cell lysates 21 …”

Application of QCM in Peptide and Protein-Based Drug Product Development

“…Modern techniques enabling observation of fiber growth include atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), experiments that detect the growth of an ensemble of fibrils by surface plasmon resonance (SPR) or dynamic light scattering (DLS). Moreover, there are also techniques employing mass spectrometry (MS) coupled with high-performance liquid chromatography (HPLC) that monitor variations in concentration of the precursor protein as a determinant of incorporation during the fibers’ growth process [ 93 , 94 ]. An alternative approach to monitoring amyloid formation, enabling, among others, measurement of the kinetics of amyloid growth via real-time monitoring of increase in fiber mass, is based on QCM technology.…”

Studies on the cross‐interaction between hIAPP and Aβ25–35 and the aggregation process in binary mixture by electrospray ionization‐ion mobility‐mass spectrometry

“…[35][36][37] Numerous studies amyloid-related peptides and proteins demonstrated that ESI-IM-MS could provide unambiguous evidence for delineating the detailed mechanism of the aggregation process and assist in developing therapeutic agents and drug evaluation. [38][39][40] In this work, ESI-IM-MS was employed to observe the nature…”