Matrix metalloproteinases (MMPs) regulate processes within the dermal and the epidermal compartments and particularly at the epidermal – dermal interface. This is of crucial importance for wound repair and also for the pathogenesis of skin cancer. One member of the MMP family - MMP-10 - gained particular interest, since it is specifically expressed at the leading edge of hyperproliferative keratinocytes in wounds and in skin tumors. The key to unravel the mechanism of MMP-10 action is the system-wide identification of its substrates under physiological and pathological conditions. To do so, we will use Terminal Amine Isotopic Labeling of Substrates (TAILS), a novel iTRAQ-based quantitative proteomic technique for the multiplex system-wide discovery of protease substrates and their cleavage sites. We will apply TAILS to identify novel bioactive MMP-10 substrates in vitro using primary keratinocytes from wild-type and MMP-10 knockout mice and from fibroblasts grown in mono- or co-culture. Thereby, we will employ 4plex-iTRAQ-TAILS to define both MMP-10 substrates and their cell type-specific origin in a single experiment. In addition, we will use TAILS to examine N-terminome changes during chemically-induced skin carcinogenesis in wild-type and MMP-10 deficient animals. This approach will allow to identify MMP-10 in vivo substrates but also to monitor the generation of neo-N-termini in wild-type controls at each important step of skin carcinogenesis in an unbiased manner. Cleavage site specificities will relate these to potential protease pools that will be defined from concomitant expression analysis using a dedicated protease microarray, the CLIP-CHIP(TM). Proteases, in particular MMPs, which are highly expressed during skin carcinogenesis, will be further analyzed for their substrates by iTRAQ-TAILS. These experiments will provide new insight into the role of proteases, including matrix metalloproteinases, in non-melanoma skin cancer development and progression.