title: Time-Resolved Analysis of Matrix Metalloproteinase Substrates in Complex Samples.
abstract: Identification of physiological substrates is the key to understanding the pleiotropic functions of matrix metalloproteinases (MMPs) in health and disease. Quantitative mass spectrometry-based proteomics has revolutionized current approaches in protease substrate discovery and helped to unravel many new MMP activities in complex biological systems. Multiplexing further extended the capabilities of these techniques and facilitated more complicated experimental designs that include multiple proteases or monitoring the activity of a single protease at more than one concentration or at multiple time points with a complex test proteome. In this chapter, we provide a protocol for time-resolved iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS), with the focus on MMP substrate identification and characterization in cell culture supernatants and introduce an automated procedure for the interpretation of time-resolved iTRAQ-TAILS datasets.
authors.affiliation: ETH Zurich, Department of Biology, Institute of Molecular Health Sciences, Otto-Stern-Weg 7, 8093, Zurich, Switzerland.
authors.forename: Pascal
authors.initials: P
authors.lastname: Schlage
authors.affiliation: ETH Zurich, Department of Biology, Institute of Molecular Health Sciences, Otto-Stern-Weg 7, 8093, Zurich, Switzerland.
authors.forename: Fabian E
authors.initials: FE
authors.lastname: Egli
authors.affiliation: ETH Zurich, Department of Biology, Institute of Molecular Health Sciences, Otto-Stern-Weg 7, 8093, Zurich, Switzerland. ulrich.aufdemkeller@biol.ethz.ch.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Auf dem Keller
mesh_terms: D002851:Chromatography, High Pressure Liquid; D055786:Gene Knockout Techniques; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D020782:Matrix Metalloproteinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D020782:Matrix Metalloproteinases
keywords: Protease; Proteomics; Substrate discovery; Time-resolved degradomics; iTRAQ-TAILS