SEC-MX: an approach to systematically study the interplay between protein assembly states and phosphorylation

Area of Science:

• Molecular Biology
• Proteomics
• Biochemistry

Background:

• Protein molecular interactions and post-translational modifications (PTMs) are co-dependent and co-regulate biological processes.
• A critical gap exists in methods for simultaneously studying protein assembly states and PTMs from the same biological sample.

Purpose of the Study:

• To introduce SEC-MX (Size Exclusion Chromatography fractions MultipleXed), a novel global quantitative method.
• To enable simultaneous characterization of PTMs and protein assembly states.
• To facilitate high-throughput, quantitative differential comparisons between biological conditions.

Main Methods:

• Combined Size Exclusion Chromatography (SEC) with PTM-enrichment techniques.
• Applied SEC-MX to HEK293 and HCT116 cell lines for proof-of-concept.
• Enabled phosphopeptide enrichment and quantitative analysis of assembly states.

Main Results:

• Generated a comprehensive dataset mapping thousands of phosphopeptides and their associated assembly states.
• Revealed intricate, previously uncharacterized relationships between phosphorylation events and protein assembly.
• Provided a foundation for generating testable hypotheses regarding protein regulation.

Conclusions:

• SEC-MX is established as a valuable tool for comprehensive protein analysis.
• The method allows exploration of protein function and regulation beyond simple abundance measurements.
• Facilitates deeper understanding of the interplay between PTMs and protein complex formation.