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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.