Identification of Protease Specificity Using Biotin-Labeled Substrates



Hiroyuki Yamamoto1, *, Syota Saito1, Yoshikazu Sawaguchi2, Michio Kimura1
1 Department of Microbiology and Molecular Cell Biology, Nihon Pharmaceutical University, 10281 Komuro, Inamachi, Kitaadachi-gun, Saitama, 362-0806, Japan
2 Department of Clinical Pharmaceutics, Nihon Pharmaceutical University, 10281 Komuro, Inamachi, Kitaadachi-gun, Saitama, 362-0806, Japan


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© 2017 Yamamoto et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Department of Microbiology and Molecular Cell Biology, Nihon Pharmaceutical University, 10281 Komuro, Inamachi, Kitaadachi-gun, Saitama, 362-0806, Japan; Tel: +81-48-721-1155; Fax: +81-48-721-6975; E-mail: yamamoto@nichiyaku.ac.jp


Abstract

Background:

Proteolysis constitutes a major post-translational modification. For example, proteases regulate the activation or inactivation of various proteins, such as enzymes, growth factors, and peptide hormones. Proteases have substrate specificity, and protease expression regulates the specific and regional activation or inactivation of several functional proteins.

Methods:

We demonstrate a novel method for determining protease specificity through the use of MALDI-TOF mass spectrometry with biotin-labeled substrates.

Results:

This method was able to determine the specificity of TPCK-trypsin, V8 protease, elastase and cyanogen bromide cleavage, and the results were similar to previous reports. In addition, the method can be used to measure crude samples, such as tumor extracts.

Conclusion:

We demonstrated that this method could identify protease specificity after simple processing, even for crude samples.

Keywords: Protease specificity, Mass spectrometry, Biotin-labeled peptides.