Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Understanding protein function requires studying multiple post-translational modifications (PTMs).
• Investigating multiple PTMs simultaneously is challenging due to high costs, time, and sample requirements.
• Existing methods often limit the analysis to a single PTM at a time.

Purpose of the Study:

• To develop a cost-effective and time-efficient protocol for simultaneous enrichment of multiple PTMs.
• To enable the study of post-translational modification crosstalk with limited sample input.
• To facilitate the identification and quantification of acetylation and succinylation PTMs.

Main Methods:

• A "one-pot" affinity enrichment protocol was developed.
• The method involves protein lysate preparation from mouse livers (SIRT5 knockout).
• Simultaneous enrichment of acetylation and succinylation was performed, followed by mass spectrometric analysis using data-independent acquisition (DIA).

Main Results:

• The protocol successfully identifies and quantifies peptides with lysine residues containing acetylation and succinylation PTMs.
• Simultaneous enrichment of two PTMs from a single sample was achieved.
• The data-independent acquisition (DIA) workflow provided comprehensive PTM-specific information, aiding in PTM site localization.

Conclusions:

• This protocol overcomes barriers in multi-PTM enrichment studies by reducing sample input, cost, and time.
• It provides a practical tool for studying PTM crosstalk.
• The DIA component enhances the ability to differentiate PTM localization isoforms.