Characterization of native protein complexes and protein isoform variation using size-fractionation-based...

Area of Science:

• Proteomics
• Cellular Biology
• Biochemistry

Background:

• Protein complexes are crucial for cellular functions.
• The role of specific protein isoforms and post-translational modifications in these complexes is not well understood.

Purpose of the Study:

• To characterize soluble protein complexes from human osteosarcoma (U2OS) cells.
• To investigate the selective participation of protein isoforms and post-translational modifications in distinct complexes.

Main Methods:

• Combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis (mass spectrometry).
• Analyzed protein complexes from 40 SEC fractions of U2OS cell lysates.
• Performed three biological replicates for statistical validation and reproducibility assessment.

Main Results:

• Identified over 71,500 peptides, 1,600 phosphosites, and 8,000 proteins, covering >50% of the U2OS proteome.
• Demonstrated reproducible SEC fractionation and identified specific protein interaction networks.
• Revealed selective association of protein isoforms and post-translational modifications with different complex size classes.
• Observed enrichment of Gene Ontology terms related to differential complex sizes.

Conclusions:

• Combined SEC/MS analysis is effective for system-wide annotation of protein complexes.
• The study provides a foundation for predicting isoform-specific protein interactions.
• Generated comprehensive SEC data integrated into the Encyclopedia of Proteome Dynamics for community access.