PhD ceremony Ms. L.M. Prély: Activity-based and targeted analyses of matrix metalloproteases
| When: | Mo 28-10-2013 at 11:00 |
PhD ceremony: Ms. L.M. Prély, 11.00 uur, Academiegebouw, Broerstraat 5, Groningen
Dissertation: Activity-based and targeted analyses of matrix metalloproteases
Promotor(s): prof. R.P.H. Bischoff, prof. A.J.M. van Oosterhout
Faculty: Mathematics and Natural Sciences
Matrix metalloproteases (MMPs) are involved in a wide range of pathological processes related to a dysfunction of their proteolytic activity. Measurement of active MMPs is thus more meaningful for the characterization of biological or pathological events than determination of the overall abundance.
The focus of the PhD project of Laurette Prély was the development of analytical techniques for monitoring MMP activity. Photoaffinity-labeling, absolute quantification by mass-spectrometry in the Selected Reaction Monitoring (SRM) mode, and activity-based enrichment techniques were investigated.
Labeling with a S1’ photoactivatable, activity-based probe and recombinant MMP-12 catalytic domain resulted in a 1:1 ratio. Furthermore, the labeled protein could be enriched with monomeric avidin beads based on the biotin residue that forms part of the probe. Characterization of the amino acid(s) implied in the covalent bond(s) with the synthetic probes is still under investigation. A targeted analysis by LC-MS/MS (SRM assay) applied to MMP-9 in Bronchoalveolar Lavage Fluid (BALF) has been developed. The developed SRM assay allows the absolute quantification of MMP-9 down to the low ng/mL level. The developed assay was adapted to MMP-12 by monitoring a different set of signature peptides.
Activity of MMP-9 is not monitored using this assay, the activity-based enrichment approach was thus combined with the MMP-9 and MMP-12 SRM assay. We further extended this methodology by monitoring peptides belonging to the pro-domain of MMP-9 as well as the main endogenous inhibitor of MMP-9, TIMP-1. Finally the limits and advantages of the developed techniques and their potential for automation are discussed.