Characterization of the Oligomeric States of Insulin in Self-Assembly and Amyloid Fibril Formation by Mass Spectrometry

Abstract

The self-assembly and aggregation of insulin molecules has been investigated by means of nanoflow electrospray mass spectrometry. Hexamers of insulin containing predominantly two, but up to four, Zn(2+) ions were observed in the gas phase when solutions at pH 4.0 were examined. At pH 3.3, in the absence of Zn(2+), dimers and tetramers are observed. Spectra obtained from solutions of insulin at millimolar concentrations at pH 2.0, conditions under which insulin is known to aggregate in solution, showed signals from a range of higher oligomers. Clusters containing up to 12 molecules could be detected in the gas phase. Hydrogen exchange measurements show that in solution these higher oligomers are in rapid equilibrium with monomeric insulin. At elevated temperatures, under conditions where insulin rapidly forms amyloid fibrils, the concentration of soluble higher oligomers was found to decrease with time yielding insoluble high molecular weight aggregates and then fibrils. The fibrils formed were examined by electron microscopy and the results show that the amorphous aggregates formed initially are converted to twisted, unbranched fibrils containing several protofilaments. Fourier transform infrared spectroscopy shows that both the soluble form of insulin and the initial aggregates are predominantly helical, but that formation of beta-sheet structure occurs simultaneously with the appearance of well-defined fibrils.

Mass spectrometry and the amyloid problem—How far can we go in the gas phase?

“…Following administration, to achieve more efficient absorption into the blood stream, the hexamers are broken down into monomers and dimers, which are prone to fibrillar self-assembly, thus limiting the therapeutic value of this approach. ESI-MS analysis of insulin in the presence of Zn 2ϩ led to the detection of monomers, dimers, tetramers, and hexamers (the latter with two to four Zn 2ϩ ions bound) [55]. The absence of odd-numbered oligomers indicated that such species were not stable entities in solution under these conditions and provided good evidence that the oligomers detected had not been generated artefactually during the ESI-MS process.…”

“…An early paper reporting the ESI-MS detection of oligomers in protein self-assembly systems focused on insulin, a 51-amino acid residue protein, which forms well-defined oligomers in its native state yet can be induced to aggregate into amyloid fibrils under nonphysiologic conditions [55]. In vivo, insulin is stored in the pancreas as a Zn 2ϩ -containing hexamer composed of three equivalent dimers, and it is this form that is used in the treatment of type 1 diabetes.…”

A polymer physics perspective on driving forces and mechanisms for protein aggregation

“…Their model predicts that macromolecular clusters should form as either thermodynamic or kinetic intermediates. Several authors have presented evidence for such clusters or oligomers for a variety of aggregation-prone IDPs [83][84][85][86][87][88][89][90][91][92]. Sizes of oligomers vary from one system to another and depend on the method of detection and the solution conditions.…”

Molecular basis for insulin fibril assembly

“…Upon fibrillation, the molecule of insulin undergoes structural changes from a predominantly ␣-helical state to a ␤-sheet rich conformation. The fibrillar ␤-sheets have been described as either parallel (11)(12)(13) or antiparallel (14)(15)(16)). An early model was based on the crystal packing of a despentapeptide insulin molecule (with residues B26-B30 removed) (4,17).…”