Chapter 12 Membrane Protease Degradomics: Proteomic Identification and Quantification of Cell Surface Protease Substrates Georgina S. Butler, Richard A. Dean, Derek Smith, and Christopher M. Overall Abstract The modification of cell surface proteins by plasma membrane and soluble proteases is important for phys- iological and pathological processes. Methods to identify shed and soluble substrates are crucial to further define the substrate repertoire, termed the substrate degradome, of individual proteases. Identifying pro- tease substrates is essential to elucidate protease function and involvement in different homeostatic and disease pathways. This characterisation is also crucial for drug target identification and validation, which would then allow the rational design of specific targeted inhibitors for therapeutic intervention. We describe two methods for identifying and quantifying shed cell surface protease targets in cultured cells utilising Isotope-Coded Affinity Tags (ICAT R  ) and Isobaric Tags for Relative and Absolute Quantifica- tion (iTRAQ TM ). As a model system to develop these techniques, we chose a cell-membrane expressed matrix metalloproteinase, MMP-14, but the concepts can be applied to proteases of other classes. By over-expression, or conversely inhibition, of a particular protease with careful selection of control con- ditions (e.g. vector or inactive protease) and differential labelling, shed proteins can be identified and quantified by mass spectrometry (MS), MS/MS fragmentation and database searching. Key words: Protease, membrane protease, matrix metalloproteinase, MMP, degradome, ICAT, iTRAQ, shedding, protease substrate identification, quantitative mass spectrometry. 1. Introduction The matrix metalloproteinases (MMPs) are a family of 23 extra- cellular zinc-dependent endopeptidases implicated in a number of physiological and pathological processes such as cancer and inflammation (1). Classically, degradation of extracellular matrix proteins has been the sole function attributed to MMPs, but in Matthew J. Peirce, Robin Wait (eds.), Membrane Proteomics: Methods and Protocols, vol. 528 C  Humana Press, a part of Springer Science+Business Media, LLC 2009 DOI 10.1007/978-1-60327-310-7 12 Springerprotocols.com 159