Development of an integrated system for activity-based profiling of matrix metallo-proteases

Abstract

Matrix metallo-proteases constitute a family of extracellular zinc-dependent endopeptidases that are involved in degradation of extracellular matrix (ECM) components and other bioactive non-ECM molecules. A plethora of studies have implicated important roles for MMPs in many diseases (including cancer and chronic inflammatory diseases) and normal biological processes. Our understanding of the complex roles of MMPs is, however, still limited, which is illustrated by the poor performance of broad-spectrum MMP inhibitors in clinical trials. The regulation of MMP activity by post-translational mechanisms is a complicating factor in MMP biology and pathology, and diminishes the effectiveness of conventional "expression-based" proteomic methods to study MMPs. The ability to profile MMPs based on functionally related activities would greatly facilitate research about their involvement in pathological processes. This thesis describes the development of a liquid chromatography-mass spectrometry-based integrated system for the selective detection of active MMPs in complex biological samples. Throughout the thesis, purified recombinant MMP-12 (catalytic domain) is used as a model enzyme. Chapter 2 demonstrates the feasibility of activity-based MMP-12 enrichment through batch extractions with an immobilized inhibitor affinity sorbent. A broad-range MMP inhibitor (PLG-NHOH) was immobilized on a Sepharose support with a ligand density of 9.8 mmol/L. The functionality of the inhibitor after immobilization was demonstrated with batch extractions of MMP-12 added to buffer, resulting in extraction yields around 97%. Quantification was done indirectly using an activity assay to determine unbound MMP-12. Active MMP-12 could also be selectively extracted when added to serum (after inactivation of the endogenous inhibitor α2-macroglobulin with trypsin) at a low level. Experiments with the endogenous inhibitors TIMP-1 and α2-macroglobulin revealed that MMP-12 extraction is strictly activity-dependent. Chapter 3 considers the automation of the extraction which is needed for on-line coupling to downstream analytical steps. Samples containing MMP-12 in buffer were extracted at different flow rates using cartridges packed with inhibitor affinity sorbent. Besides faster extractions and a reduced number of manual manipulations, higher extraction yields (98.9% - 99.3%) were obtained over the whole flow rate range compared to batch extractions. Application of the method to synovial fluid from a rheumatoid arthritis patient followed by gelatin-zymography revealed a strong enrichment of distinct MMPs from this biological sample that were not clearly visible in the original sample. The use of an auto-sampler and a solid-phase extraction (SPE) workstation allowed full automation of the extraction procedure. MMP-12 extractions were optimized, showing that ligand density is an important factor with a clear extraction yield optimum around 4 to 10 mmol/L. Conditioning of the Sepharose affinity sorbent for 1 week prior to use resulted in a further increase in extraction yield. Under optimal conditions, an extraction yield of 99.5% was reached with a cartridge contact time of only 13 s for MMP-12. The efficacy of the extraction method for activity-based MMP profiling was further improved by the use of a broad-spectrum MMP inhibitor with nmol/L affinity (TAPI-2), which resulted in increased extraction yields for all tested MMPs. Extraction yields ranging from 98.8% to 99.8% were obtained for MMP-1, -7, -8, -10, -12, and -13, while for MMP-9 (full length and catalytic domain) extraction yields of 96.1% and 98.4% were reached (all at a cartridge contact time of 19 s). This effective enrichment of MMPs illustrates the possibility to enrich from dilute samples with low levels of endogenous MMPs. In Chapter 4, we investigated the chemical modification (acetylation) of immobilized trypsin (on a Sepharose and a polystyrene support) for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was obtained with modified-trypsin beads with a contact time of only 4 sec, while corresponding unmodified-trypsin beads gave incomplete digestion. The digestion rate of myoglobin, a protein known to be rather resistant to proteolysis, was not altered by acetylating trypsin and required a buffer containing 35% acetonitrile to obtain complete digestion. The use of acetylated-trypsin beads led to fewer interfering tryptic autolysis products, indicating an increased stability of this modified enzyme. Importantly, the modification did not affect trypsin's substrate specificity, as the peptide map of myoglobin was not altered upon acetylation of immobilized trypsin. Kinetic digestion experiments in solution with low-molecular-weight substrates and with cytochrome c confirmed the increased catalytic efficiency (lower KM and higher kcat and increased resistance to autolysis of trypsin upon acetylation. Because of the increased trypsin activity, the digestion rate is enhanced, which facilitates on-line digestion of low-abundance proteins with higher yields and in less time. These are favourable properties of the modified trypsin reactor and should make it a valuable tool in automated protein analysis systems. Chapter 5 deals with the implementation of the extraction and the digestion cartridge into an integrated system for automated, activity-based profiling of MMPs. Enrichment was followed by on-line digestion in an immobilized acetylated trypsin reactor. Hyphenation of the sample pre-treatment steps to a nanoLC-MS system was achieved by loading tryptic MMP-12 peptides on a reversed-phase trap column, followed by backflush elution to a 50-µm reversed-phase silica-based monolith capillary column coupled to a nanoESI interface and an ion trap mass spectrometer. The use of non-ionic surfactants in the sample pre-treatment steps resulted in better sensitivity, probably because on-line digestion efficiency is improved and non-specific adsorption is suppressed. The completely automated method is able to analyse a sample every 75 min. Spiking of MMP-12 at 4 pmol into 500 µL urine resulted in selective detection of tryptic MMP-12 peptides with high intensities. At sub-pmol MMP-12 levels the signal dropped strongly, but some tryptic MMP-12 peptides were still detected at 0.25 pmol in 100 µL injections (from buffer) with good signal-to-noise ratios. Though the developed system has not been extensively tested with biological samples to detect active endogenous MMPs, several aspects should likely be improved in order to achieve this. One important limiting factor in obtaining low or sub-fmol sensitivity is probably the rate of digestion in the trypsin reactor. Despite the relatively high concentration of immobilized trypsin and the chemical modification, the digestion rate is still limited by the protein substrate concentration (due to Michaelis-Menten kinetics). The use of support materials with less diffusional transport limitations such as monolithic materials in both the enrichment and the digestion step will likely result in improved digestion rates at low MMP concentrations. Captured MMPs can be eluted from a monolithic extraction cartridge in a smaller volume and thus be digested at higher concentration in digestion reactor, resulting in improved digestion kinetics. Reduced diffusional transport limitation in the trypsin reactor may also contribute to a further improvement of the digestion kinetics at low MMP concentrations. Digestion kinetics may also be further improved through the investigation of other types of chemical modifications of immobilized trypsin. Hydrophilic protein modifications generally result in an enhanced thermal stability and may permit the use of elevated digestion temperatures to achieve faster digestion kinetics. A decreased binding of hydrophobic peptides to immobilized trypsin, resulting in higher peptide yields and a lower carry-over is an additional advantage of hydrophilic modifications. Chapter 5 demonstrated a positive effect of non-ionic surfactant additions to the extraction and digestion buffers on the mass spectrometric MMP-12 peptide signals. Yet, higher levels of these surfactants have an adverse effect on the effective capacity (due to overloading) of the reversed-phase trap column and will pollute the ESI interface. The option to include an ion-exchange step, either on- or off-line to wash away high levels of surfactant prior to reversed-phase trapping, should thus be a subject of further research. Care must to be taken, however, not to introduce too many system steps, which would result in high system complexity accompanied by low robustness. Validation (LOD, LOQ, day-to-day and intra-day variation) of the integrated analytical system should be performed before application to clinical studies, once the system has been further optimized. The aspect of quantification of detected MMPs should also be addressed in future research. To correct for different yields of the different steps, ideally an internal standard with a known concentration, added to the biological sample, should be used. This can be achieved by using isotopically labelled MMPs (produced by expression in a host organism which is grown on 15N-enriched medium). To broaden the applicability of the developed system to a more diverse group of metallo-proteases, the use of affinity sorbents with a cocktail of immobilized metallo-protease inhibitors targeting different types of enzymes can be investigated. Through the use of affinity sorbents targeting completely different protein classes (of low abundance), the developed system can be potentially applied in a wide range of studies. The requirement for different elution conditions with each type of affinity sorbent affects the on-line coupling with the digestion reactor and is the main step which needs to be evaluated before the system can be used to analyse other protein classes. Digestion reactors with immobilized proteases, other than trypsin, may be required to ensure elution-digestion compatibility.