Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry...

Area of Science:

• Proteomics and Systems Biology
• Molecular and Cellular Biology
• Biochemistry

Background:

• Protein-protein interactions (PPIs) are crucial for cellular functions, and their alterations are linked to diseases.
• Existing methods for studying PPIs quantitatively can be costly and time-consuming, limiting large-scale applications.
• Robust methods are needed to analyze PPI changes induced by genetic variations or external stimuli.

Purpose of the Study:

• To develop and validate a quantitative, label-free approach for characterizing changes in protein-protein interactions.
• To assess the impact of specific mutations and drug treatments on protein complex composition.
• To establish a scalable pipeline for systems biology studies of PPI networks.

Main Methods:

• Affinity purification coupled with mass spectrometry (AP-MS) was employed.
• Data-independent mass spectrometry acquisition (sequential window acquisition of all theoretical spectra, SWATH) was integrated with AP.
• An automated data extraction and statistical analysis pipeline was implemented to score interaction modulation.

Main Results:

• The developed AP-SWATH method successfully characterized PPI changes.
• Specific examples include alterations induced by an HSP90 inhibitor (NVP-AUY922) and melanoma-associated mutations in CDK4.
• The approach demonstrated robustness and sensitivity for label-free quantitative analysis.

Conclusions:

• AP-SWATH provides a robust and scalable label-free method for quantifying changes in protein-protein interactions.
• This technique facilitates systems biology approaches to understand complex cellular processes and disease mechanisms.
• The automated pipeline enables efficient analysis of large-scale PPI studies.