title: pCAP-based peptide substrates: the new tool in the box of tyrosine phosphatase assays.
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.
authors.affiliation: Division of Cellular Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Stanford
authors.affiliation: 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.
authors.forename: Divya
authors.initials: D
authors.lastname: Krishnamurthy
authors.affiliation: Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Rhushikesh A
authors.initials: RA
authors.lastname: Kulkarni
authors.affiliation: 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.
authors.forename: Caitlin E
authors.initials: CE
authors.lastname: Karver
authors.affiliation: Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Eveline
authors.initials: E
authors.lastname: Bruenger
authors.affiliation: Division of Cellular Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA.
authors.forename: Logan M
authors.initials: LM
authors.lastname: Walker
authors.affiliation: Conrad Prebys Center for Chemical Genomics, Sanford|Burnham Medical Research Institute, La Jolla, CA 92037, USA.
authors.forename: Chen-Ting
authors.initials: CT
authors.lastname: Ma
authors.affiliation: Conrad Prebys Center for Chemical Genomics, Sanford|Burnham Medical Research Institute, La Jolla, CA 92037, USA.
authors.forename: Thomas D Y
authors.initials: TD
authors.lastname: Chung
authors.affiliation: Conrad Prebys Center for Chemical Genomics, Sanford|Burnham Medical Research Institute, La Jolla, CA 92037, USA.
authors.forename: Eduard
authors.initials: E
authors.lastname: Sergienko
authors.affiliation: Division of Cellular Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. Electronic address: nunzio@liai.org.
authors.forename: Nunzio
authors.initials: N
authors.lastname: Bottini
authors.affiliation: Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA. Electronic address: amy.barrios@utah.edu.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Barrios
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D001681:Biological Assay / Q000379:methods*; D003374:Coumarins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D010755:Organophosphates / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D010755:Organophosphates; D010455:Peptides; D017027:Protein Tyrosine Phosphatases; D000409:Alanine
keywords: Enzyme activity gel; Fluorogenic enzyme substrates; High-throughput screening; Multiply phosphorylated peptides; Peptide synthesis