Abstract
Differential proteolytic processing, for example by matrix metalloproteases (MMPs), has been recognized as an important hallmark in numerous pathological conditions. One crucial challenge in the present studies of proteases is system-wide identification of endogenous biological substrates. In this chapter, we highlight a robust method for the identification of bioactive substrates and their sites of MMP cleavage, as well as by other proteases and peptidases, in a system-wide manner. This approach enriches for putative protein N-termini by removal of internal peptides using a charge reversal strategy. In addition, this straightforward method can be used in combination with gel-based pre-separation of proteins to allow better estimation of the molecular weight of the identified cleavage product of a given bioactive substrate.
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Acknowledgment
This study was funded by a Marie Curie Fellowship for Career Development (PIIF-GA-2012-329622 GlycoMarker to Z.W.L.), Deutsche Forschungsgemeinschaft (SCHI 871/2 and SCHI 871/5, SCHI 871/6, GR 1748/6, INST 39/900-1 and SFB850-Project B8 to O.S.), European Research Council (ERC-2011- StG 282111-ProteaSys to O.S.), and the Excellence Initiative of the German Federal and State Governments (EXC 294, BIOSS to O.S.).
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Lai, Z.W., Schilling, O. (2017). Identification of Protease Cleavage Sites by Charge-Based Enrichment of Protein N-Termini. 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_10
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DOI: https://doi.org/10.1007/978-1-4939-6863-3_10
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