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Protein Sci. 2018 Mar;27(3):584-594. doi: 10.1002/pro.3352. Epub 2017 Dec 8.

Global substrate specificity profiling of post-translational modifying enzymes.

Author information

1
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California.
2
Pharmaceutical Sciences and Pharmacogenomics Graduate Program, University of California, San Francisco, San Francisco, California.
3
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California.

Abstract

Enzymes that modify the proteome, referred to as post-translational modifying (PTM) enzymes, are central regulators of cellular signaling. Determining the substrate specificity of PTM enzymes is a critical step in unraveling their biological functions both in normal physiological processes and in disease states. Advances in peptide chemistry over the last century have enabled the rapid generation of peptide libraries for querying substrate recognition by PTM enzymes. In this article, we highlight various peptide-based approaches for analysis of PTM enzyme substrate specificity. We focus on the application of these technologies to proteases and also discuss specific examples in which they have been used to uncover the substrate specificity of other types of PTM enzymes, such as kinases. In particular, we highlight our multiplex substrate profiling by mass spectrometry (MSP-MS) assay, which uses a rationally designed, physicochemically diverse library of tetradecapeptides. We show how this method has been applied to PTM enzymes to uncover biological function, and guide substrate and inhibitor design. We also briefly discuss how this technique can be combined with other methods to gain a systems-level understanding of PTM enzyme regulation and function.

KEYWORDS:

kinases; mass spectrometry; peptide libraries; peptide synthesis; post-translation modifying enzymes; proteases; substrate specificity

PMID:
29168252
PMCID:
PMC5818756
[Available on 2019-03-01]
DOI:
10.1002/pro.3352

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