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Molecular & Cellular Proteomics 7:2215-2228, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach^*,S

Danmei Xu^,,¶, Naoko Suenaga^,,||, Mariola J. Edelmann^**, Rafael Fridman, Ruth J. Muschel^, and Benedikt M. Kessler^**,¶¶

From the Radiation Oncology and Biology, Medical Science Division, Churchill Hospital, University of Oxford, Oxford OX3 7LJ, United Kingdom, ^** Central Proteomics Facility, Henry Wellcome Building for Molecular Physiology, Department of Clinical Medicine, Roosevelt Drive, University of Oxford, Oxford OX3 7BN, United Kingdom, and Department of Pathology, School of Medicine, Wayne State University, Detroit, Michigan 48201

Matrix metalloproteinase-9 (MMP-9) is implicated in tumor metastasis as well as a variety of inflammatory and pathological processes. Although many substrates for MMP-9, including components of the extracellular matrix, soluble mediators such as chemokines, and cell surface molecules have been identified, we undertook a more comprehensive proteomics-based approach to identify new substrates to further understand how MMP-9 might contribute to tumor metastasis. Previous proteomics approaches to identify protease substrates have depended upon differential labeling of each sample. Instead we used a label-free quantitative proteomics approach based on ultraperformance LC-ESI-high/low collision energy MS. Conditioned medium from a human metastatic prostate cancer cell line, PC-3ML, in which MMP-9 had been down-regulated by RNA interference was compared with that from the parental cells. From more than 200 proteins identified, 69 showed significant alteration in levels after depletion of the protease (>±2-fold), suggesting that they might be candidate substrates. Levels of six of these (amyloid-β precursor protein, collagen VI, leukemia inhibitory factor, neuropilin-1, prostate cancer cell-derived growth factor (PCDGF), and protease nexin-1 (PN-1)) were tested in the conditioned media by immunoblotting. There was a strong correlation between results by ultraperformance LC-ESI-high/low collision energy MS and by immunoblotting giving credence to the label-free approach. Further information about MMP-9 cleavage was obtained by comparison of the peptide coverage of collagen VI in the presence and absence of MMP-9 showing increased sensitivity of the C- and N-terminal globular regions over the helical regions. Susceptibility of PN-1 and leukemia inhibitory factor to MMP-9 degradation was confirmed by in vitro incubation of the recombinant proteins with recombinant MMP-9. The MMP-9 cleavage sites in PN-1 were sequenced. This study provides a new label-free method for degradomics cell-based screening leading to the identification of a series of proteins whose levels are affected by MMP-9, some of which are clearly direct substrates for MMP-9 and become candidates for involvement in metastasis.

^¶¶ Supported by an MRC New Investigator Award. To whom correspondence may be addressed. Tel.: 44-1865-287-799; Fax: 44-1865-287-787; E-mail: bmk{at}ccmp.ox.ac.uk

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