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Oligopeptide Competition Assay for Phosphorylation Site Determination

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Fluorogenic peptide substrates for serine and threonine phosphatases.

Fengtian Xue¹, Christopher T Seto

¹Department of Chemistry, Brown University, Providence, Rhode Island 02912, USA.

Organic Letters

|April 3, 2010

Summary

A novel fluorescent assay detects Ser/Thr protein phosphatases by monitoring the release of a fluorescent reporter upon substrate hydrolysis. This method shows sequence selectivity, aiding in profiling important phosphatases.

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Defining Substrate Specificities for Lipase and Phospholipase Candidates

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Published on: November 23, 2016

Area of Science:

Biochemistry
Enzymology
Assay Development

Background:

Serine/threonine (Ser/Thr) protein phosphatases are crucial regulators of cellular signaling.
Dysregulation of phosphatase activity is implicated in various diseases, making them important drug targets.
Existing assays may lack the sensitivity or specificity for comprehensive phosphatase profiling.

Purpose of the Study:

To develop a new, sensitive fluorescent assay for Ser/Thr protein phosphatases.
To characterize the sequence selectivity of the developed assay using different phosphatase enzymes.
To evaluate the assay's potential for profiling medicinally relevant phosphatases.

Main Methods:

A novel fluorescent reporter system was designed, which is released upon hydrolysis of a phosphoSer residue.
The assay mechanism involves cyclization of an N-terminal carbamate after phosphoSer hydrolysis.
Enzyme kinetics and substrate specificity were assessed using peptide substrates and purified phosphatases, including alkaline phosphatase (ALP), bacteriophage lambda protein phosphatase (lambda-PPase), and vaccinia H1 related phosphatase (VHR).

Main Results:

A functional fluorescent assay for Ser/Thr protein phosphatases was successfully developed.
The assay demonstrated sequence selectivity when tested against ALP, lambda-PPase, and VHR.
Specific peptide substrates elicited differential responses, highlighting the assay's ability to distinguish between phosphatase activities.

Conclusions:

The developed fluorescent assay is a valuable tool for detecting Ser/Thr protein phosphatase activity.
The assay's sequence selectivity enables detailed profiling of substrate specificities.
This assay holds promise for advancing research into the roles of phosphatases in health and disease.