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Using Peptide Arrays to Profile Phosphatase Activity in Cell Lysates
Lindsey C Szymczak1, Daniel J Sykora2, Milan Mrksich1,2
1Department of Chemistry, Northwestern University, Evanston, IL 60208, USA.
Chemistry (Weinheim an Der Bergstrasse, Germany)
|November 7, 2019
Summary
Global phosphorylation regulation is poorly understood. This study used SAMDI-MS to reveal significant differences in protein phosphatase activity across cell lines, highlighting their crucial role in controlling phosphorylation.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Phosphorylation is a key post-translational modification regulating cellular processes.
- The mechanisms and regulation of global protein phosphorylation are not fully understood.
Purpose of the Study:
- To profile global phosphatase activity in mammalian cell lysates.
- To investigate differences in phosphatase activity on serine, threonine, and tyrosine substrates.
Main Methods:
- Utilized self-assembled monolayers on gold for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS).
- Employed three phosphorylated peptide arrays for high-throughput screening.
- Analyzed cell lysates from five distinct mammalian cell lines.
Main Results:
- Identified significant variations in protein phosphatase activity across different cell lines.
- Observed distinct phosphatase activities towards phosphoserine, phosphothreonine, and phosphotyrosine substrates.
- Demonstrated substantial differences in global phosphatase activity.
Conclusions:
- Protein phosphatases play a more significant role in regulating global protein phosphorylation than previously recognized.
- Phosphatase activity is substrate-specific and varies across cell types.
- This work provides new insights into the complex regulation of phosphorylation.
