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Methods. 2014 Jan 15;65(2):165-74. doi: 10.1016/j.ymeth.2013.07.022. Epub 2013 Jul 22.

pCAP-based peptide substrates: the new tool in the box of tyrosine phosphatase assays.

Author information

1
Division of Cellular Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA.
2
Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA; Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
3
Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
4
Conrad Prebys Center for Chemical Genomics, Sanford|Burnham Medical Research Institute, La Jolla, CA 92037, USA.
5
Division of Cellular Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. Electronic address: nunzio@liai.org.
6
Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA. Electronic address: amy.barrios@utah.edu.

Abstract

Robust, facile high throughput assays based on non-peptidic probes are available to detect the enzyme activity of protein tyrosine phosphatases. However, these assays cannot replace the use of peptide-based probes in many applications; for example when a closer mimic of the physiological target is desired or in substrate profiling expeditions. Phosphotyrosine peptides are often used in these assays, but their use is complicated by either poor sensitivity or the need for indirect detection methods, among other pitfalls. Novel peptide-based probes for protein tyrosine phosphatases are needed to replace phosphotyrosine peptides and accelerate the field of tyrosine phosphatase substrate profiling. Here we review a type of peptidic probe for tyrosine phosphatases, which is based on the incorporation of the phosphotyrosine-mimic phosphocoumaryl amino propionic acid (pCAP) into peptides. The resulting fluorogenic pCAP peptides are dephosphorylated by tyrosine phosphatases with similar efficiency as the homologous phosphotyrosine peptides. pCAP peptides outperform phosphotyrosine peptides, providing an assay that is as robust, sensitive and facile as the non-peptidic fluorogenic probes on the market. Finally the use of pCAP can expand the range of phosphatase assays, facilitating the investigation of multiphosphorylated peptides and providing an in-gel assay for phosphatase activity.

KEYWORDS:

Enzyme activity gel; Fluorogenic enzyme substrates; High-throughput screening; Multiply phosphorylated peptides; Peptide synthesis

PMID:
23886911
PMCID:
PMC3899110
DOI:
10.1016/j.ymeth.2013.07.022
[Indexed for MEDLINE]
Free PMC Article
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