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Metalloproteases play a complex role in tumor progression. Proteomic approaches to identify the array of substrates of a given metalloprotease (degradome), can help to unveil its role in tumor growth and metastasis. Here we describe a proteomic screening using two complementary approaches, 2D-DIGE and SDS-PAGE LC-MS/MS SILAC analysis, to identify new substrates of the metalloprotease ADAMTS1. MCF7 mammary cancer cells were engineered to conditionally express ADAMTS1 using the Tet-Off system. Cells grown in conditions were the expression is induced or switched off were differentially labelled by SILAC. Glycoproteins released to the conditioned media of each of the two cell cultures were purified by lectin affinity chromatography. Samples of the two conditions were compared by the 2D-DIGE methodology, or pooled and run on a 1D SDS-PAGE gel followed by RP-LC-MS/MS. The DIGE approach led to the identification, among others, of Semaphorin 3C (S3C) as a new potential substrate of ADAMTS1, together with Thrombospondin-1, a known substrate of the protease. A total of 3163 and 2697different peptides, from 749 and 728 individual proteins, were identified and quantified, respectively, in two complementary SILAC screenings, in which isotopic labelling was swapped. A number of known substrates of ADAMTS1 were identified in the analysis, such as Thrombospondin-1 and Dystroglycan. In addition several new candidate substrates of the protease were identified, including S3C. Processing of S3C by ADAMTS1 was further validated and characterized, showing the protease-dependent release of a 95 kDa S3C fragment into the medium. Cell migration assays showed that this S3C fragment is able to promote migration of breast cancer cells.. Semaphorins, first described as axon guidance factors, have also been related to tumor progression. Our results show that metalloproteases can regulate cell migration through Semaphorin processing , thus contributing to the metastatic process.