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2-DIMENSIONAL DEGRADOMICS AS A POWERFUL TOOL TO IDENTIFY NOVEL GELATINASE B/MMP-9 SUBSTRATES Cauwe B, Martens E, Proost P, Opdenakker G. Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium email: benedicte.cauwe@rega.kuleuven.be With more than 20.000 entries in the PubMed database, matrix metalloproteinases (MMPs) are a family of enzymes with many key functions in biology and pathology. Although MMPs first came into the spotlights as extracellular matrix (ECM) wreckers, recent evidence shows that they also degrade other molecules, including intracellular matrix (ICM) proteins. In contrast with the large collection of secreted (cytokines, protein hormones, chemokines,…) and membrane-associated MMP substrates (adhesion molecules, receptors,…) that have been described and characterized in detail, only a dozen intracellular MMP substrates were identified so far. Gelatinase B/matrix metalloproteinase-9 is the most complex family member in terms of structure and function and takes the honour of about half of all MMP literature entries. MMP-9 has been studied as a molecular target in cancer and in acute and chronic inflammatory diseases. We used a degradomic approach to characterize the intracellular ‘degradome’ or substrate repertoire of MMP-9 on cytosol of THP-1 cells. In the first dimension, anion and cation exchange chromatography separated THP-1 cytosolic proteins by their net charge and/or isoelectric point (pI). Each elution fraction was then concentrated, dialyzed and incubated in the absence or presence of MMP-9. In the second dimension, digested and undigested fractions were separated by molecular weight by SDS-PAGE. Disappearance of proteins after incubation with MMP-9 or appearance of novel fragment bands point to potential substrate proteins. The differential bands were identified by LC-MS/MS after in-gel tryptic digests. In this way, we were able to isolate hundreds of potential intracellular MMP-9 substrates, ~70 of which were identified by MS/MS. As a proof-of-principle, some of these proteins were already described as MMP-9 substrates in the literature. Novel candidate substrates were confirmed by western blot analysis, detecting MMP-9-generated fragments in THP-1 cytosol, or by cleavage of the purified or recombinant substrate in vitro. This unprejudiced, systematic approach has a number of advantages in comparison with other methods. First, the substantial loading capacity of ion exchange columns made it possible to start with a high protein amount. This, together with the concentration of the ion exchange fractions, allowed us to identify low-abundant substrates that might be missed starting from a small unfractionated protein sample. Second, by incubating fractionated cytosol with MMP-9 less bias occurs by high-abundant, high-affinity substrates that ‘consume’ the protease and mask lower-affinity, but still potential physiological substrates, in a complex mixture. Finally, 2-dimensional degradomics can be extended to other cell types, culture media, tissues or body fluids, turning it into a powerful tool for high-throughput identification of novel MMP substrates.