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Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry
Published on: April 4, 2018
HPLC-based quantification of in vitro N-terminal acetylation
Rune H Evjenth1, Petra Van Damme, Kris Gevaert
1Department of Molecular Biology, University of Bergen, Bergen, Norway.
Methods in Molecular Biology (Clifton, N.J.)
|February 6, 2013
Summary
Researchers developed a new in vitro method to study protein N-terminal acetylation. This technique helps determine the specificity and kinetics of N-terminal acetyltransferases (NATs), advancing our understanding of this crucial cellular process.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Protein N-terminal acetylation is a common post-translational modification in eukaryotic organisms.
- This process is catalyzed by a family of enzymes known as N-terminal acetyltransferases (NATs).
- The precise substrate specificities and catalytic mechanisms of various NATs remain incompletely elucidated.
Purpose of the Study:
- To develop and validate a quantitative in vitro method for assessing NAT activity.
- To enable the characterization of NAT substrate specificity.
- To determine kinetic parameters of NAT-catalyzed acetylation reactions.
Main Methods:
- Development of an in vitro acetylation assay using oligopeptide substrates.
- Quantification of acetylation using reverse-phase High-Performance Liquid Chromatography (HPLC).
Main Results:
- The described method allows for quantitative measurement of in vitro N-terminal acetylation.
- The assay is capable of determining the substrate specificity of different NATs.
- Kinetic parameters for NAT activity can be reliably measured using this approach.
Conclusions:
- This novel HPLC-based method provides a robust tool for studying N-terminal acetylation.
- It facilitates a deeper understanding of NAT function, specificity, and kinetics.
- The method can aid in the characterization of NATs and their roles in biological processes.
