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.

摘要

Matrix metalloproteinases (MMPs) network with other biological molecules toudmaintain the extracellular matrix (ECM) in normal physiology and performuddifferent roles. Understanding and assigning specific role to each of 24udmembers of these endoproteinases is impeded because of lack of specificudand efficient detection methods in biological samples. Moreover, MMP-basedudanti-cancer drug development has also been challenged because, currently,udthere is no robust methodology to distinguish the inactive pro-enzymes,udactive enzymes or those complexed with endogenous inhibitors in biologicaludspecimens. The objective of this project is to develop a chemical proteomicsudstrategy based on Matrix assisted laser desorption ionization tandem massudspectrometry (MALDI-MS/MS) to help identify and discriminate the variousudMMP forms. Firstly, a triazine dye-based ligand immobilized onudchromatography beads was utilized to assess whether it binds toudrecombinant human MMPs (rhMMPs). The results highlighted that the ligandudinteracts with latent forms of MMPs in agreement with the literature.udSecondly, the potential of the ligand was assessed using MALDI-MS/MSudbased methodology in in vitro cancer models. Cell line culture supernatantsudwere used in amounts to emulate the availability of tumour biopsies in clinicaludsettings. The MS/MS spectral peaks specific to MMPs (MMP-2 and MMP-ud14), and two endogenous inhibitors TIMP-1 and TIMP-2 were found in affinityudchromatography eluates of cell culture supernatants with higher Mascotudscores for the latter. While western blot detected MMP-2 in cell extracts,udMALDI-MS/MS did not detect MMPs because of amounts below the limit ofuddetection (LOD) of the instrument. Although the ligand was found to beudinteracting with MMPs and detergent-free salt elution buffers improvedudMALDI analysis, recovery of MMPs from biological samples was sub-optimal.udThe dye ligand was observed to bind other enzymes and despite variousudstrategies to reduce non-specific binding of proteins or enable selectiveudelution did not improve MMP enrichment. Further work using methodologyuddescribed in this study is required after scaling up the MMP amounts inudbiological specimen and to resolve the issue of non-specific binding ofudproteins to the ligand by understanding its structure.