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dc.contributor.advisorBrodbelt, Jennifer S.
dc.creatorRobotham, Scott Allen
dc.date.accessioned2017-06-13T22:17:16Z
dc.date.available2017-06-13T22:17:16Z
dc.date.issued2015-05
dc.date.submittedMay 2015
dc.identifierdoi:10.15781/T20R9M911
dc.identifier.urihttp://hdl.handle.net/2152/47211
dc.description.abstractAlthough in silico database search methods remain more popular for shotgun proteomics methods, de novo sequencing offers the ability to identify proteins lacking sequenced genomes and ones with subtle splice variants or truncations. Ultraviolet photodissociation (UVPD) of peptides derivatized by selective attachment of a chromophore at the N-terminus generated characteristic series of y ions. The UVPD spectra of the chromophore-labelled peptides were simplified and thus amenable to de novo sequencing. E.coli lysates were modified by the use of carbamylation and the attachment of a UV chromophore to the N-terminus of digested peptides. UVPD of the resulting peptides generated clean sets of y ions. A novel de novo algorithm, UVnovo, afforded high confidence identification of thousands of peptides from an E. coli lysate and allowed UVPD/CID paired spectra to be searched. E.coli lysate peptides were analyzed in alternating scans by UVPD and CID on the same precursors to generate paired UVPD/CID spectra. UVnovo enabled sequence tag de novo sequencing of peptides in order to find matching sequences from a database. Ultimately the UVnovo functioned as a standalone de novo sequencing program or a hybrid de novo/database search program. In an effort to better characterize the fragmentation pathways promoted by ultraviolet photoexcitation in comparison to CID, six adrenocorticotropic hormone (ACTH) peptides in a range of charge states were subjected to 266 nm ultraviolet photodissociation (UVPD), 193 nm UVPD, and CID. While both UVPD and CID led to preferential cleavage of the Y-S bond for all ACTH peptides (except ACTH (1-39)), UVPD was far less dependent on charge state and location of basic sites for the production of C-terminal and N-terminal ions.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.subjectMass spectrometry
dc.subjectUltraviolet photodissociation
dc.subjectDe novo sequencing
dc.subjectPeptide characterization
dc.subjectProteomics
dc.titleDe novo sequencing and peptide characterization via ultraviolet photodissociation mass spectrometry
dc.typeThesis
dc.date.updated2017-06-13T22:17:16Z
dc.contributor.committeeMemberCrooks, Richard M
dc.contributor.committeeMemberEllington, Andrew
dc.contributor.committeeMemberShear, Jason B
dc.contributor.committeeMemberWebb, Lauren
dc.description.departmentChemistry
thesis.degree.departmentChemistry
thesis.degree.disciplineChemistry
thesis.degree.grantorThe University of Texas at Austin
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy
dc.type.materialtext


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