The computational analysis of post-translational modifications

The post-translational modification (PTMs) of proteins presents a means to increase the proteome size and diversity of an organism through the inclusion of structural elements not encoded at the sequence-level alone. Their erroneous inclusion or exclusion has been linked to a variety of diseases and disorders thus their characterisation has the potential to present viable drug targets. The proliferation of newer high-throughput methods, such as mass spectrometry, to identify such modifications has led to a rapid increase in the number of databases and tools to display and analyse such vast amounts of data effectively. This study covers the development of one such tool; PTM Browser, and the construction of the underlying database that it is based upon. This new database was initially seeded with annotations from the Swiss-Prot and Phospho.ELM resources. The initial database of PTMs was then expanded to include a large repertoire of previously unannotated proteins for a selection of topical species (e.g. Danio rerio and Tetraodon nigroviridis). Orthologue assignments have also been added to the database – to allow for queries to be performed regarding the conservation of modifications between homologous proteins. The PTM Browser tool allows for a full exploration of this new database of PTMs – with a special focus on allowing users to identify modifications that are both shared between and are specific to particular species. This tool is freely available for non-commercial use at the following URL: http://www.ptmbrowser.org. An analysis is presented on the conservation of modifications between members of the tumour suppressor family, p53, using this new tool. This tool has also been used to analysis the conservation of modifications between super-kingdoms and Eukaryote species

Chemical studies into the amino acids present in latent fingermarks

The aim of this research was to carry out investigations into the amino acid chemistry of latent fingermarks. Amino acids are the primary targets of chemical visualisation methods for fingermarks deposited on porous substrates. Two novel amino acid sensitive reagents for the detection of latent fingermarks were formulated and evaluated. Analytical chemistry methodology was developed and subsequently applied to examine the amino acid content of latent fingermarks deposited on paper

Host cell factors in ovarian cancer influencing efficacy of oncolytic aenovirus dl922-947

PhDAdenoviral gene therapy holds great potential for cancer treatment, but is limited by a lack of efficient vectors. dl922-947, an E1A CR2-deleted adenovirus, replicates selectively within and lyses cancer cells. It is believed that its selectivity depends upon abnormalities in the cell cycle regulatory Rb pathway and subsequent G1/S checkpoint, observed in 90% of human cancers. The cytotoxic efficacy of dl922-947 is greater than that of wild-type adenovirus and dl1520 (Onyx-015). Nevertheless, sensitivity to dl922-947 varies widely among ovarian cancer cell lines, despite similar infectivity. My work aimed to identify host cell factors that influence cytotoxicity and which could be potential biomarkers in clinical trials. Surprisingly, comparison of ovarian cancer lines indicated that cytotoxicity correlated poorly with infectivity, replication and virion production. Immunoblotting suggested correlation between sensitivity to dl922-947 and overexpression of p21, p27, Cyclin D, cdk4 and p16. Subsequent experiments confirmed a role for p21 in dl922-947 cytotoxic activity. In vitro and in vivo, Hct116 p21+/+ cells were significantly more sensitive to dl922-947 than matched p21-/- cells. p21 knock-down by siRNA in TOV21G and IGROV-1 cells reduced dl922-947 cytotoxicity, whilst re-expression in ACP-WAF1 cells increased activity. p21 expression was also greater in sensitive transformed TOSE cells compared to resistant normal IOSE25 cells. Results suggest that p21 promotes dl922-947 activity by stabilising Cyclin D thus promoting cell cycle progression. Comparative microarray analysis in TOSE1, 4 and IOSE25 cells and in MRC5 and MRC5-VA cells suggested determinants of dl922-947 activity beyond the Rb pathway, which may also prove valuable biomarkers. Moreover, pathways and processes emerged, correlating with sensitivity, and meriting future investigation. Together, my results suggest that a cellular environment conducive for dl922-947 function includes mediators of proliferative capacity, amongst which p21 plays a role in enhancing activity of the viru

Negative Ion Electron Capture Dissociation (niECD): A Novel Tandem Mass Spectrometric Technique.

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) are powerful tandem mass spectrometry (MS/MS) techniques for biomolecular structural elucidation. However, one drawback of ECD/ETD is that they require multiply positively charged precursor ions, possibly precluding analysis of acidic molecules such as phospho-, sulfopeptides, and sialylated glycopeptides. Electron attachment to anions appears unlikely due to Coulomb repulsion. However, we found that such an intriguing reaction is indeed feasible within a narrow energy range (3.5-6.5 eV). The resulting charge-increased radicals further undergo dissociation analogous to ECD/ETD, thus constituting a novel MS/MS technique that we termed negative ion electron capture dissociation (niECD). niECD of phospho- and sulfopeptides yields predictable c’/z•-type backbone fragments without loss of phosphoric acid or sulfonate. Fragmentation pattern similarities between niECD and ECD indicate that niECD proceeds through a mechanism related to that of ECD. We proposed that gas-phase zwitterionic structures are necessary for successful niECD and that a positive charge is required to serve as the electron capture site, or to promote electron capture. N-terminal acetylation, which should reduce the probability of zwitterion formation, results in decreased niECD efficiency and introduction of fixed positive charge tags, which should promote zwitterion formation, enables niECD of peptides which could not undergo niECD in their unmodified forms. niECD efficiency also decreases with decreasing zwitterion propensity for five sets of synthetic peptides, further supporting the zwitterion mechanism. niECD was further applied to peptide chains bound by natural disulfide bonds or disulfide-containing cross-linkers. niECD of disulfide-linked peptides again results in very similar fragmentation patterns as those from ECD. S-S bond cleavage constitutes the preferred fragmentation pathway, producing characteristic fragments, which allow rapid detection of disulfide-linked peptides. Analogous to cation ECD, niECD of both N-linked and O-linked sialylated glycopeptides, which are readily deprotonated in negative ion mode, exhibits peptide backbone fragmentation with retention of labile glycans. Overall, the research presented in this thesis contributes to an increased understanding of the mechanism and utility of niECD, thereby allowing this unique approach to be developed into a valuable analytical tool for structural analysis of important biological samples.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/109024/1/ningwang_1.pd