CHARACTERIZATION OF POLYUBIQUITIN CHAINS BY MASS SPECTROMETRY

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dc.contributor.advisorFenselau, Catherineen_US
dc.contributor.authorLee, Amanda Elizabethen_US
dc.contributor.departmentChemistryen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2016-09-03T05:35:09Z
dc.date.available2016-09-03T05:35:09Z
dc.date.issued2016en_US
dc.description.abstractThe work outlined in this dissertation will allow biochemists and cellular biologists to characterize polyubiquitin chains involved in their cellular environment by following a facile mass spectrometric based workflow. The characterization of polyubiquitin chains has been of interest since their discovery in 1984. The profound effects of ubiquitination on the movement and processing of cellular proteins depend exclusively on the structures of mono and polyubiquitin modifications anchored or unanchored on the protein within the cellular environment. However, structure-function studies have been hindered by the difficulty in identifying complex chain structures due to limited instrument capabilities of the past. Genetic mutations or reiterative immunoprecipitations have been used previously to characterize the polyubiquitin chains, but their tedium makes it difficult to study a broad ubiquitinome. Top-down and middle-out mass spectral based proteomic studies have been reported for polyubiquitin and have had success in characterizing parts of the chain, but no method to date has been successful at differentiating all theoretical ubiquitin chain isomers (ubiquitin chain lengths from dimer to tetramer alone have 1340 possible isomers). The workflow presented here can identify chain length, topology and linkages present using a chromatographic-time-scale compatible, LC-MS/MS based workflow. To accomplish this feat, the strategy had to exploit the most recent advances in top-down mass spectrometry. This included the most advanced electron transfer dissociation (ETD) activation and sensitivity for large masses from the orbitrap Fusion Lumos. The spectral interpretation had to be done manually with the aid of a graphical interface to assign mass shifts because of a lack of software capable to interpret fragmentation across isopeptide linkages. However, the method outlined can be applied to any mass spectral based system granted it results in extensive fragmentation across the polyubiquitin chain; making this method adaptable to future advances in the field.en_US
dc.identifierhttps://doi.org/10.13016/M25B8P
dc.identifier.urihttp://hdl.handle.net/1903/18550
dc.language.isoenen_US
dc.subject.pqcontrolledAnalytical chemistryen_US
dc.subject.pqcontrolledChemistryen_US
dc.subject.pqcontrolledBiochemistryen_US
dc.subject.pquncontrolledbranched proteinsen_US
dc.subject.pquncontrolledelectron transfer dissociationen_US
dc.subject.pquncontrolledmiddle-outen_US
dc.subject.pquncontrolledProteomicsen_US
dc.subject.pquncontrolledtop-downen_US
dc.subject.pquncontrolledtopologyen_US
dc.titleCHARACTERIZATION OF POLYUBIQUITIN CHAINS BY MASS SPECTROMETRYen_US
dc.typeDissertationen_US

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