title: Time-resolved analysis of the matrix metalloproteinase 10 substrate degradome.
abstract: Proteolysis is an irreversible post-translational modification that affects intra- and intercellular communication by modulating the activity of bioactive mediators. Key to understanding protease function is the system-wide identification of cleavage events and their dynamics in physiological contexts. Despite recent advances in mass spectrometry-based proteomics for high-throughput substrate screening, current approaches suffer from high false positive rates and only capture single states of protease activity. Here, we present a workflow based on multiplexed terminal amine isotopic labeling of substrates for time-resolved substrate degradomics in complex proteomes. This approach significantly enhances confidence in substrate identification and categorizes cleavage events by specificity and structural accessibility of the cleavage site. We demonstrate concomitant quantification of cleavage site spanning peptides and neo-N and/or neo-C termini to estimate relative ratios of noncleaved and cleaved forms of substrate proteins. By applying this strategy to dissect the matrix metalloproteinase 10 (MMP10) substrate degradome in fibroblast secretomes, we identified the extracellular matrix protein ADAMTS-like protein 1 (ADAMTSL1) as a direct MMP10 substrate and revealed MMP10-dependent ectodomain shedding of platelet-derived growth factor receptor alpha (PDGFRα) as well as sequential processing of type I collagen. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000503.
authors.affiliation: ETH Zurich, Department of Biology, Institute of Molecular Health Sciences, Schafmattstr. 22, 8093 Zurich, Switzerland;
authors.forename: Pascal
authors.initials: P
authors.lastname: Schlage
authors.forename: Fabian E
authors.initials: FE
authors.lastname: Egli
authors.forename: Paolo
authors.initials: P
authors.lastname: Nanni
authors.forename: Lauren W
authors.initials: LW
authors.lastname: Wang
authors.forename: Jayachandran N
authors.initials: JN
authors.lastname: Kizhakkedathu
authors.forename: Suneel S
authors.initials: SS
authors.lastname: Apte
authors.forename: Ulrich
authors.initials: U
authors.lastname: auf dem Keller
mesh_terms: D000818:Animals; D041702:BALB 3T3 Cells; D020134:Catalytic Domain; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D007553:Isotope Labeling / Q000379:methods*; D053506:Matrix Metalloproteinase 10 / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D059748:Proteolysis; D020543:Proteome / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D010446:Peptide Fragments; D020543:Proteome; D053506:Matrix Metalloproteinase 10