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Ion exchange chromatography separates charged molecules from a solution by reversibly exchanging them with mobile, or 'active', ions associated with the oppositely charged stationary phase. This method can be used to separate ions, soften and deionize water, and purify solutions. The polymers comprising the ion-exchange column are high-molecular-weight and chemically stable polymers, crosslinked to be porous and essentially insoluble. They are also functionalized with either acidic or...

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A Strong Cation Exchange Chromatography Protocol for Examining N-Terminal Proteoforms.

Esperanza Fernández^1,2, Annelies Bogaert^1,2, Evy Timmerman^1,2,3

¹VIB Center for Medical Biotechnology, Ghent, Belgium.

Methods in Molecular Biology (Clifton, N.J.)

|May 6, 2022

Summary

This study presents a proteomics method to enrich N-terminally acetylated peptides. This technique aids in identifying new protein forms and quantifying acetylation levels in eukaryotes.

Keywords:

N-terminal acetylation N-terminal acetyltransferases N-terminomics

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Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

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Area of Science:

Biochemistry
Proteomics
Molecular Biology

Background:

N-terminal acetylation is a crucial post-translational modification in eukaryotes, influencing protein function and stability.
Identifying N-terminally acetylated peptides is vital for understanding protein synthesis and regulation.
Existing methods for N-terminal peptide analysis can be challenging and lack efficiency.

Purpose of the Study:

To develop and present a robust bottom-up proteomics protocol for the selective enrichment of N-terminally acetylated peptides.
To enable the identification of novel N-terminal proteoforms and the quantification of N-terminal acetylation.
To provide a valuable tool for studying the roles of N-terminal acetyltransferases (NATs).

Main Methods:

Utilizing strong cation exchange chromatography (SCX) for peptide enrichment.
Depleting internal tryptic peptides by exploiting their retention on SCX.
Analyzing the non-retained fraction for enriched N-terminally acetylated/blocked peptides.

Main Results:

Successful enrichment of N-terminally acetylated peptides from complex proteome digests.
Demonstration of the protocol's ability to identify previously unknown N-terminal proteoforms.
Quantification of the degree of N-terminal protein acetylation is feasible with this method.

Conclusions:

The developed SCX-based proteomics protocol offers an efficient strategy for N-terminal peptide enrichment.
This method significantly advances the study of N-terminal acetylation and its biological implications.
The protocol facilitates comprehensive analysis of protein N-termini, aiding in proteome-wide acetylation studies.