Study of the Stability of Insulin Oligomers in the Presence and Absence of Zn(Ii) Using Mass Spectrometry

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Date of Award

Fall 2012

Abstract

Insulin is a hormone protein essential to the regulation of blood sugar levels. It is mainly stored in the body as a hexamer in the presence of zinc ion, which induces an inactive form with long-term stability.1 The main focus of this research is the assessment of the effects of zinc ions on the formation and stability of insulin oligomers, including the ability of oligomers to transfer from solution to the gas phase. The self-assembly, aggregation and stability of insulin oligomers with and without zinc ions has been investigated by ion mobility-mass spectrometry (IMMS). Studying the conformation and stability of insulin oligomers enables better understanding of the hormones reactivity and opens up the potential for the improvement of drugs. Using electrospray ionization - ion mobility - mass spectrometry (ESI-IM-MS) the different charge states and oligomers of insulin were separated and identified. Different oligomers of insulin were observed to exist depending on the solvent, pH, and different concentrations of the zinc ion and insulin. Using collision-induced dissociation, the insulin oligomers' relative stabilities and order of formation have been studied. A solution made from 50% acetonitrile, 47.5% water, 1% acetic acid and 1.5% of 5:1 and 10:1 insulin: zinc chloride solution of 5.0 � 10 -4 M concentration was found to give the best result. This solution showed the presence of monomers ranging from +1 to +4 charge states, dimers from +2 to +4, trimers from +3 to +6, tetramers from +4 to +8, pentamers from +5 to +8 and hexamers from +6 to +10. Upon collisional activation, insulin oligomers with none, one and two zinc ions were observed to fragment in approximately the same way, with zinc ions retained by the larger aggregate oligomers, with evidence of greater stability for oligomers bound to two zinc ions. For example, collisional activation of the 7+ tetramer, 7+ tetramer-Zn(II), and 7+ tetramer-2Zn(II) exhibited the formation of the 5+ trimer and 2+ monomer with the zinc remaining attached to the 5+ trimer. The 7+ tetramer-2Zn2+ also exhibited a greater stability than the 7+ tetramer and 7+ tetramer-Zn2+. The same experiments were performed on a range of oligomers to try and see which oligomers are stabilized by the zinc ions.

Advisor

Laurence Angel

Subject Categories

Chemistry | Physical Sciences and Mathematics

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