Browsing University of Bradford eTheses by Subject "Tandem Mass Spectrometry"
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Proteomic Profiling of Pro and Active Matrix Metalloproteinases using Tandem Mass Spectrometry. Optimization of Affinity Chromatography and nHPLC-MALDI-MS/MS for Proteomic discrimination of Matrix Metalloproteinases in pre-clinical Cancer Model.Matrix metalloproteinases (MMPs) network with other biological molecules to maintain the extracellular matrix (ECM) in normal physiology and perform different roles. Understanding and assigning specific role to each of 24 members of these endoproteinases is impeded because of lack of specific and efficient detection methods in biological samples. Moreover, MMP-based anti-cancer drug development has also been challenged because, currently, there is no robust methodology to distinguish the inactive pro-enzymes, active enzymes or those complexed with endogenous inhibitors in biological specimens. The objective of this project is to develop a chemical proteomics strategy based on Matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) to help identify and discriminate the various MMP forms. Firstly, a triazine dye-based ligand immobilized on chromatography beads was utilized to assess whether it binds to recombinant human MMPs (rhMMPs). The results highlighted that the ligand interacts with latent forms of MMPs in agreement with the literature. Secondly, the potential of the ligand was assessed using MALDI-MS/MS based methodology in in vitro cancer models. Cell line culture supernatants were used in amounts to emulate the availability of tumour biopsies in clinical settings. The MS/MS spectral peaks specific to MMPs (MMP-2 and MMP- 14), and two endogenous inhibitors TIMP-1 and TIMP-2 were found in affinity chromatography eluates of cell culture supernatants with higher Mascot scores for the latter. While western blot detected MMP-2 in cell extracts, MALDI-MS/MS did not detect MMPs because of amounts below the limit of detection (LOD) of the instrument. Although the ligand was found to be interacting with MMPs and detergent-free salt elution buffers improved MALDI analysis, recovery of MMPs from biological samples was sub-optimal. The dye ligand was observed to bind other enzymes and despite various strategies to reduce non-specific binding of proteins or enable selective elution did not improve MMP enrichment. Further work using methodology described in this study is required after scaling up the MMP amounts in biological specimen and to resolve the issue of non-specific binding of proteins to the ligand by understanding its structure.