Identification of Protease Specificity Using Biotin-Labeled Substrates

Yamamoto, Hiroyuki; Saito, Syota; Sawaguchi, Yoshikazu; Kimura, Michio

Identification of Protease Specificity Using Biotin-Labeled Substrates

Hiroyuki Yamamoto^{1, *}, 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

Article Information

Identifiers and Pagination:

Year: 2017
Volume: 11
First Page: 27
Last Page: 35
Publisher ID: TOBIOCJ-11-27
DOI: 10.2174/1874091X01711010027

Article History:

Received Date: 19/08/2016
Revision Received Date: 14/02/2017
Acceptance Date: 17/03/2017
Electronic publication date: 21/04/2017
Collection year: 2017

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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

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.

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RESEARCH ARTICLE

Identification of Protease Specificity Using Biotin-Labeled Substrates

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Abstract

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Conclusion:

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The Open Biochemistry Journal

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