Development of novel mass spectrometric methods for identifying HOCl-induced modifications to proteins

Protein oxidation is thought to contribute to a number of inflammatory diseases, hence the development of sensitive and specific analytical techniques to detect oxidative PTMs (oxPTMs) in biological samples is highly desirable. Precursor ion scanning for fragment ions of oxidized amino acid residues was investigated as a label-free MS approach to mapping specific oxPTMs in a complex mixture of proteins. Using HOCl-oxidized lysozyme as a model system, it was found that the immonium ions of oxidized tyrosine and tryptophan formed in MS(2) analysis could not be used as diagnostic ions, owing to the occurrence of isobaric fragment ions from unmodified peptides. Using a double quadrupole linear ion trap mass spectrometer, precursor ion scanning was combined with detection of MS(3) fragment ions from the immonium ions and collisionally-activated decomposition peptide sequencing to achieve selectivity for the oxPTMs. For chlorotyrosine, the immonium ion at 170.1 m/z fragmented to yield diagnostic ions at 153.1, 134.1, and 125.1 m/z, and the hydroxytyrosine immonium ion at 152.1 m/z gave diagnostic ions at 135.1 and 107.1 m/z. Selective MS(3) fragment ions were also identified for 2-hydroxytryptophan and 5-hydroxytryptophan. The method was used successfully to map these oxPTMs in a mixture of nine proteins that had been treated with HOCl, thereby demonstrating its potential for application to complex biological samples.

The anti-inflammatory action of tetracyclines

For six decades tetracyclines have been successfully used for their broad spectrum antibiotic effects. However, non-antibiotic effects of tetracyclines have been reported. The anti-inflammatory effects of tetracycline drugs have been investigated in the context of a range of inflammatory diseases including sepsis and a number of neurodegenerative diseases. This thesis investigates the effects of a range of clinically important tetracyclines (oxytetracycline, doxycycline, minocycline and tigecycline) on the ability of the J774.2 cell line to produce nitric oxide when stimulated with the bacterial cell wall component, LPS. The proteome of J774.2 cells was analysed in response to LPS stimulation (1 µg/ml) with and without prior treatment with minocycline (50µg/ml), this allows the unbiased analysis of the cellular proteome in response to minocycline and LPS, protein spots of interest were excised and identified by nano-electrospray ionisation-linear ion trap mass spectroscopy. All of the tetracyclines that were investigated inhibited LPS-induced nitric oxide production in a dose dependent manner and this was due to the inhibition of inducible nitric oxide synthase expression. This is the first report to show that tigecycline inhibits inducible nitric oxide expression and nitric oxide production. Using two-dimensional gel electrophoresis and total protein staining eleven proteins were identified as being modulated by LPS. Of these eleven proteins; expression of some, but not all was modulated when the cells received a prior treatment with minocycline suggesting that minocycline does not completely block LPS-induced macrophage activation but probably specifically acts on particular inflammatory signaling pathways in macrophages. Three protein spots with a similar molecular weight but different pI values identified in this proteomic study were identified as ATP synthase ß chain. These different protein spots probably correspond to different phosphorylation states of the protein, suggesting that minocycline affects the balance of protein kinase and protein phosphatase activity in the immune response.