Mass spectrometry has become an important tool for analysis of protein complexes. This study utilizes electrospray ionization (ESI) coupled to a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) to analyze noncovalent complexes in the gas phase. Binding of cucurbit[7]uril (CB7) to intact bovine insulin and the B-chain of insulin was investigated. Competition experiments involving the B-chain and a mutant B-chain were performed to probe the solution-phase binding site. Electron capture dissociation (ECD) of CB7 complexed to intact insulin and to the B-chain, produced a series of peptidic fragments of insulin in complex with CB7. Analysis of these fragments allowed the determination of the apparent gas-phase binding site, which appears different than the proposed solution-phase binding-site. These studies thus suggest that CB7 migrates when the complex is transferred from solution to gas phase. The results of this study caution against using ECD-MS as a stand-alone structural probe of solutionphase binding.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/32467 |
| Date | 19 July 2012 |
| Creators | Heath, Brittany |
| Contributors | Jockusch, Rebecca A. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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