Generation of High-Quality Succinyl Spectral Libraries for Improved Proteome-Wide Succinylome Analysis Using...

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Posttranslational modifications (PTMs) like lysine succinylation are vital for protein function and cellular signaling.
• Analyzing succinylation is challenging, but mass spectrometry, especially data-independent acquisition (DIA), has improved proteome-wide analysis.
• DIA data processing requires spectral libraries for accurate identification and quantification of low-abundance peptides.

Purpose of the Study:

• To describe a workflow for building data-dependent acquisition (DDA) posttranslational modification (PTM) spectral libraries.
• To demonstrate the use of these libraries for analyzing DIA succinylome data.
• To enhance the accuracy and visualization of succinylated peptide quantification.

Main Methods:

• Generation of DDA PTM spectral libraries using Spectronaut, SpectroMine, and MSFragger.
• Utilizing spectral libraries within Skyline for DIA data analysis.
• Quantitative analysis of succinylome data from Sirtuin-5 knockout vs. wild-type mouse brains.

Main Results:

• Successful construction of DDA PTM spectral libraries.
• Accurate quantification and visualization of succinylated peptides in DIA data.
• Improved identification reproducibility and quantification accuracy for succinylome analysis.

Conclusions:

• DDA PTM spectral libraries are essential for robust DIA-based succinylome analysis.
• The described workflow enhances the ability to study lysine succinylation.
• This approach facilitates a deeper understanding of the role of succinylation in biological processes.