Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Overview

This study presents a novel mass spectrometric workflow for quantifying protein post-translational modifications, specifically acetylation and succinylation. The method enhances measurement accuracy by determining site-specific occupancy or stoichiometry of these modifications.

Key Study Components

Area of Science

• Biochemistry
• Mass Spectrometry
• Proteomics

Background

• Post-translational modifications play a crucial role in protein function.
• Understanding acetylation and succinylation is vital for studying reversible protein regulation.
• Traditional methods may lack precision in quantifying these modifications.
• This study aims to improve the accuracy of stoichiometry measurements.

Purpose of Study

• To develop a workflow for accurate quantification of protein acetylation and succinylation.
• To identify which lysine sites are most modified and their impact on protein function.
• To provide a method that can analyze peptides with multiple lysine residues.

Methods Used

• Ratiometric analysis of endogenous modifications.
• Quantitative chemical acylation using stable isotope-labeled anhydrides.
• Data-independent acquisition mass spectrometry.
• Preparation of multiple replicates for accuracy.

Main Results

• The method allows for precise determination of site-level stoichiometry.
• It reveals the proportion of modified versus unmodified lysine residues.
• Higher measurement accuracy compared to traditional methods.
• Identifies key acetylation and succinylation sites affecting protein function.

Conclusions

• This workflow significantly enhances the quantification of protein modifications.
• It provides insights into the regulation of protein function through acetylation and succinylation.
• The technique is applicable to a wide range of proteomic studies.

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