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. / Shaukat Khanam Memorial Cancer Hospital and Research
Centre, Pakistan and University of Bradford
| Identifer | oai:union.ndltd.org:BRADFORD/oai:bradscholars.brad.ac.uk:10454/5752 |
| Date | January 2012 |
| Creators | Saleem, Saira |
| Contributors | Sutton, Chris W., Gill, Jason H. |
| Publisher | University of Bradford, Institute of Cancer Therapeutics |
| Source Sets | Bradford Scholars |
| Language | English |
| Detected Language | English |
| Type | Thesis, doctoral, PhD |
| Rights | <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/"><img alt="Creative Commons License" style="border-width:0" src="http://i.creativecommons.org/l/by-nc-nd/3.0/88x31.png" /></a><br />The University of Bradford theses are licenced under a <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/">Creative Commons Licence</a>. |
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