Quantification and Identification of Post-Translational Modifications Using Modern Proteomics Approaches.

Area of Science:

• Biochemistry
• Proteomics
• Cellular Biology

Background:

• Post-translational modifications (PTMs) are crucial for dynamic cellular responses.
• Lysine residues are targets for various PTMs, including acetylation, methylation, and ubiquitination.
• Efficient identification of PTMs is essential for understanding cellular signaling.

Purpose of the Study:

• To describe a detailed protocol for PTM analysis.
• To enable high-throughput, label-free quantification of protein acetylation.
• To compare acetylation changes across different conditions.

Main Methods:

• Tissue homogenization and proteolytic protein digestion.
• Immunoaffinity enrichment of lysine-acetylated peptides.
• High-resolution, data-independent acquisition (DIA) mass spectrometry.

Main Results:

• Identification of lysine-acetylated peptides.
• Quantification of relative changes in acetylation.
• Establishment of a robust PTM analysis workflow.

Conclusions:

• The described protocol provides an efficient method for PTM analysis.
• This technique facilitates high-throughput, label-free quantification of acetylation.
• The workflow is valuable for studying cellular responses to environmental stimuli.