| dc.contributor.author | Jakubec, Philip | |
| dc.date.accessioned | 2021-12-21T12:23:22Z | |
| dc.date.available | 2021-12-21T12:23:22Z | |
| dc.date.issued | 2021-12-21T12:23:22Z | |
| dc.identifier.uri | http://hdl.handle.net/10222/81139 | |
| dc.description | The research presented in this thesis is in the field of proteomics, the study of proteins. These sensitive molecules can be analyzed using liquid chromatography mass spectrometry (LC-MS). These biological systems require specific preparation and handling protocols to preserve their structures and detergent based workflows are commonly used. Detergents denature the proteins structure aiding in solubilizing the molecules. This allows for the complex mixture of all the proteins in a cell to be separated, isolated, and enriched in abundance. These are measures and protocols that were developed to increase the proteins MS detection signals. Sodium dodecyl sulfate (SDS) is a commonly used detergent in proteomic experiments. Transmembrane electrophoresis (TME) is platform developed to rapidly remove SDS from proteins. The research presented here describes how the TME platform was improved using an active-cooling (AC), water-circulating system for continuous heat removal. This mitigates proteins thermally denaturing and allowed for the application of higher currents for increased SDS depletion rates. Furthermore, an in-situ TME protease digestion protocol was developed to retrieve any thermally denature proteins that retained in the system as aggregated pellets. The new active-cooled transmembrane electrophoresis (AC-TME) platform was applied to a bottom-up proteomics LC-MS workflow. | en_US |
| dc.description.abstract | Transmembrane electrophoresis (TME) is an electrokinetic method designed to remove sodium dodecyl sulfate (SDS) from protein samples prior to mass spectrometry analysis. Proteins in solution are stored behind dialysis membranes while the electromotive force of an applied electric field drives the charged surfactant off the proteins and across the membrane. For optimal MS analysis, SDS must be depleted below 10 ppm. An actively cooled TME (AC-TME) system is presented that employs water-circulating tubes to mitigate Joule heat build up and to maximize the rate of SDS depletion. To further remove the potential for protein loss through TME purification, an in-situ TME trypsin digestion protocol was also developed and demonstrated here. The improved protein purification device and protocol presented removes one of the major bottlenecks in proteome analysis, enabling the use of SDS-based workflows in a robust and high throughput format, for proteomic LC-MS analysis. | en_US |
| dc.language.iso | en | en_US |
| dc.subject | Proteomics | en_US |
| dc.subject | Purification | en_US |
| dc.subject | Transmembrane Electrophoresis | en_US |
| dc.title | Electrokinetic Removal of Sodium Dodecyl Sulfate from Proteins for Mass Spectrometry Analysis | en_US |
| dc.date.defence | 2021-12-07 | |
| dc.contributor.department | Department of Chemistry | en_US |
| dc.contributor.degree | Master of Science | en_US |
| dc.contributor.external-examiner | n/a | en_US |
| dc.contributor.graduate-coordinator | Dr. Peng Zhang | en_US |
| dc.contributor.thesis-reader | Dr. Heather Andreas | en_US |
| dc.contributor.thesis-reader | Dr. Michael Freund | en_US |
| dc.contributor.thesis-supervisor | Dr. Alan Doucette | en_US |
| dc.contributor.ethics-approval | Not Applicable | en_US |
| dc.contributor.manuscripts | Yes | en_US |
| dc.contributor.copyright-release | No | en_US |
