Endogenous protein interactomes resolved through immunoprecipitation-coupled quantitative proteomics in cell lines

Area of Science:

• Proteomics
• Molecular Biology
• Biochemistry

Background:

• Identifying protein-protein interactions is crucial for understanding cellular mechanisms.
• Endogenous immunoprecipitation (IP) is a key technique for studying these interactions in a native context.

Purpose of the Study:

• To describe an adaptable immunoprecipitation protocol for identifying endogenous protein interactions.
• To detail a quantitative proteomics workflow for unbiased interactome discovery.

Main Methods:

• Utilized antibody-based immunoprecipitation of target proteins.
• Employed quantitative proteomics including tryptic digestion, Tandem Mass Tag (TMT) labeling, and peptide fractionation.
• Performed liquid chromatography-mass spectrometry (LC-MS) for peptide identification and quantification.
• Included computational and statistical analysis for data interpretation.

Main Results:

• Successfully established an adaptable IP protocol for endogenous proteins.
• Developed a quantitative proteomics workflow enabling unbiased interactome identification.
• The protocol facilitates comprehensive analysis of protein complexes.

Conclusions:

• The described IP and quantitative proteomics workflow provides a powerful and adaptable method for discovering protein interactomes.
• This approach enhances the biological relevance of protein interaction studies.