Updated project metadata. Secreted and cell-surface proteases are major mediators of extracellular matrix remodelling, but their mechanisms and regulatory impact are poorly understood. We developed a mass spectrometry approach using cell free ECM produced by mouse Balb/c 3T3 cells in vitro to identify fibronectin as a novel substrate of the secreted metalloprotease ADAMTS16. ADAMTS16 cleaves fibronectin between its (I)5 and (I)6 modules, releasing the N-terminal 30kDa heparin-binding domain essential for fibronectin assembly.