A basic phosphoproteomic-DIA workflow integrating precise quantification of phosphosites in systems biology

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Phosphorylation is a key post-translational modification (PTM) regulating protein function.
• Advancements in mass spectrometry have enabled large-scale phosphoproteomic studies.
• Optimized protocols are needed to integrate phosphoproteomics with data-independent acquisition (DIA) for enhanced analysis.

Purpose of the Study:

• To describe an optimized Phos-DIA protocol for robust phosphoproteomic analysis.
• To provide a detailed workflow from sample preparation to bioinformatic analysis.
• To facilitate both small-scale and large-scale quantification of phosphorylation events.

Main Methods:

• Development and optimization of the Phos-DIA protocol.
• Integration of phosphoproteomics with data-independent acquisition (DIA).
• Comprehensive bioinformatic analysis pipeline for site-specific phosphorylation events.

Main Results:

• A robust and feasible Phos-DIA protocol is presented.
• The protocol allows for the measurement of tens of thousands of site-specific phosphorylation events.
• The method is applicable to both small-scale and large-scale sample quantification.

Conclusions:

• The optimized Phos-DIA protocol enhances phosphoproteomic analysis.
• This method supports systems biology and systems medicine research through large-scale quantification.
• The protocol offers practical considerations for experimental configuration and data analysis.