CHARACTERIZATION OF POLYUBIQUITIN CHAINS BY MASS SPECTROMETRY

The 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.

INVESTIGATION BY MASS SPECTROMETRY OF THE UBIQUITOME AND PROTEIN CARGO OF EXOSOMES DERIVED FROM MYELOID-DERIVED SUPPRESSOR CELLS

Exosomes released by myeloid-derived suppressor cells (MDSC) are 30 nm in diameter extracellular vesicles that have been shown to carry biologically active proteins as well as ubiquitin molecules. Ubiquitin is known to have many functions, including involvement in the formation of exosomes, although the exact role is highly contested. In the study reported here, the proteome and ubiquitome of MDSC exosomes has been investigated by bottom-up proteomics techniques. This report identifies more than 1000 proteins contained in the MDSC exosome cargo and 489 sites of ubiquitination in more than 300 ubiquitinated proteins based on recognition of glycinylglycine tagged peptides without antibody enrichment. This has allowed extensive chemical and biological characterization of the ubiquitinated cohort compared to that of the entire protein cargo to support hypotheses on the role of ubiquitin in exosomes.

Appropriateness of Largemouth Bass as a Model Species for Detection of Endocrine Dissruption

Intersex in largemouth bass (Micropterus salmoides) has been correlated with regional anthropogenic activity, but has not been causally linked to environmental factors. Four groups of hatchery-reared largemouth bass (LMB) and fathead minnows (FHM) of varying ages and sex were exposed to aqueous poultry litter mixtures, 17β- estradiol (E2), and controls. Water samples were analyzed for estrogens through liquid chromatography tandem mass spectrometry and estrogenicity through the bioluminescent yeast estrogen screen assay. Fish plasma was analyzed for the egg yolk protein vitellogenin (Vtg) using enzyme–linked immunosorbent assay and gonad tissue was examined histologically for enumeration of testicular oocytes (TO). Water chemistry revealed typical E2 conversion to Estrone with subsequent decay over the exposure periods. A modest prevalence of TO (9.4%) was detected with no apparent treatment effect. While significant Vtg induction was found in E2 exposed FHM, minimal Vtg induction was found in male LMB. Despite field findings of intersex in male LMB, this species may be poorly suited for laboratory investigations into endocrine disruption.