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.
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Acknowledgments
A special thanks goes to S. Werner (ETH Zurich) for continuous support of our research. We want to thank Paolo Nanni, Tobias Kockmann, and the whole proteomics team of the Functional Genomics Center Zurich (FGCZ) for excellent support in mass spectrometry. This work was funded by grants from the Swiss National Science Foundation (31003A_140726 and 31003A_163216), the European Commission (Marie Curie International Reintegration Grant; FP7-PEOPLE- 2010-RG/SkiNterminomics) and the Novartis Foundation for Medical-Biological Research to U.a.d.K., and by funds from the ETH Zurich.
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Schlage, P., Egli, F.E., auf dem Keller, U. (2017). Time-Resolved Analysis of Matrix Metalloproteinase Substrates in Complex Samples. In: Galea, C. (eds) Matrix Metalloproteases. Methods in Molecular Biology, vol 1579. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6863-3_9
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DOI: https://doi.org/10.1007/978-1-4939-6863-3_9
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