Dataset Information

SILAC-iTRAQ-TAILS - Monitoring matrix metalloproteinase activity at the epidermal-dermal interface by SILAC-iTRAQ-TAILS

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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Dataset's files

Source:

			Action	DRS
	20131219_05_GluC_PO.mgf	Mgf
	20131219_05_GluC_PO.mzXML	Mzxml
	20131219_05_GluC_PO.raw	Raw
	20131219_06_GluC_NPO.mgf	Mgf
	20131219_06_GluC_NPO.mzXML	Mzxml

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Publications

Monitoring matrix metalloproteinase activity at the epidermal-dermal interface by SILAC-iTRAQ-TAILS.

Schlage Pascal P Kockmann Tobias T Kizhakkedathu Jayachandran N JN auf dem Keller Ulrich U

Proteomics 20150515 14

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 may 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 ...[more]

PMID: 25871442

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