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dc.contributor.advisorCotton, Robin W.
dc.contributor.authorSamuel, Jacob Matthew
dc.date.accessioned2019-01-10T16:09:21Z
dc.date.available2019-01-10T16:09:21Z
dc.date.issued2018
dc.identifier.urihttps://hdl.handle.net/2144/33039
dc.description.abstractProtamine, a set of small basic proteins (P1 and P2), play a key role in compacting and protecting the DNA in sperm. As such, the structure of how P1 and P2 bind to DNA and potentially themselves and each other is of interest to several fields including forensics. In forensic DNA analysis, protamine binding of DNA is taken advantage of in the “differential extraction” procedure in which a sample that contains sperm and non-sperm cells can have DNA from the two different cell types separated and extracted at different points thus preventing a mixture of DNA. A key component of this greater structure and what makes the differential extraction functional are the disulfide bonds formed by protamine. So as a first step to elucidating the protamine-DNA complex, methods to analyze human protamine via 2D-Liquid Chromatography-Mass Spectrometry (2D-LC-MS) were developed in the hopes they could be used for disulfide bond mapping. Methods and multiple strategies for digestion, 2D-LC-MS were investigated and developed using chum salmon protamine. Digestion strategies were developed for Chymotrypsin and Lys-C, Trypsin or Arg-C with incubation times and substrate:enzyme mass ratios optimized. Various “trap and elute” 2D-chromatography configuration were tested for analysis intact and digested protein. Using H2O with 2% NH4OH as the loading mobile phase and H2O and Acetonitrile both with 0.5% formic acid as the eluting mobile phases with the first dimension column being an HLB (Hydrophilic Lipophilic Balance) 2.1 x 30 mm column and the second dimension being an C18 2.1 x 100 mm was found to produce the highest signal.en_US
dc.language.isoen_USen_US
dc.subjectBiochemistryen_US
dc.subjectProtamineen_US
dc.subjectDisulfide bondsen_US
dc.subjectMass spectrometryen_US
dc.subjectMultidimensional liquid chromatographyen_US
dc.titleDevelopment of methdos for the analysis of human protamine via 2D LC-MS/MSen_US
dc.typeThesis/Dissertationen_US
dc.date.updated2018-10-25T19:03:59Z
etd.degree.nameMaster of Scienceen_US
etd.degree.levelmastersen_US
etd.degree.disciplineBiomedical Forensic Sciencesen_US
etd.degree.grantorBoston Universityen_US


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