SILAC-iTRAQ-TAILS - Monitoring matrix metalloproteinase activity at the epidermal-dermal interface by SILAC-iTRAQ-TAILS
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ABSTRACT: Secreted proteases act on interstitial tissue secretomes released from multiple cell types. Thus, substrate proteins might be part of higher molecular complexes constituted by many proteins with diverse and potentially unknown cellular origin. In cell culture these might be reconstituted by mixing native secretomes from different cell types prior to incubation with a test protease. Although current degradomics techniques could identify novel substrate proteins in these complexes, all information on the cellular origin would be lost. To address this limitation we combined iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (iTRAQ-TAILS) with stable isotope labeling by amino acids in cell culture (SILAC) to assign proteins to a specific cell type by MS1- and their cleavage by MS2-based quantification in the same experiment. We demonstrate the power of our newly established workflow by monitoring matrix metalloproteinase (MMP) 10-dependent cleavages in mixtures from heavy labeled fibroblast and light labeled keratinocyte sectretomes. This analysis correctly assigned extracellular matrix components, such as laminins and collagens, to their respective cellular origins and revealed their processing in an MMP10-dependent manner. Hence, our newly devised degradomics workflow facilitates deeper insights into protease activity in complex intercellular compartments like the epidermal-dermal interface by integrating multiple modes of quantification with positional proteomics.
INSTRUMENT(S): Q Exactive
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Keratinocyte, Fibroblast
SUBMITTER: Ulrich auf dem Keller
LAB HEAD: Ulrich auf dem Keller
PROVIDER: PXD001643 | Pride | 2015-05-11
REPOSITORIES: Pride
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