Exposing the subunit diversity within protein complexes: a mass spectrometry approach

Area of Science:

• Proteomics
• Structural Biology
• Biochemistry

Background:

• Protein complexes are crucial for biological functions, but their complexity is underestimated.
• Subunit diversity, including post-translational modifications and alternative splicing, is key to complex regulation.
• A comprehensive understanding requires methods that can identify and characterize these diverse subunits.

Purpose of the Study:

• To develop and validate a mass spectrometry-based approach for exposing protein subunit heterogeneity in complexes.
• To provide a detailed description of the methodology for analyzing protein variants within complexes.
• To demonstrate the application of this method on the human COP9 signalosome complex.

Main Methods:

• Protein complexes are denatured and subunits separated using a monolithic column.
• A Triversa NanoMate robot splits the flow for parallel analysis.
• On-line electrospray ionization quadrupole time-of-flight (ESI-QToF) mass spectrometry measures intact protein mass.
• Subsequent proteomic analysis in a 96-well plate identifies subunit sequence.
• Accurate mass measurements are correlated with sequence identity to reveal heterogeneity.

Main Results:

• The method successfully exposes the array of protein variants comprising protein complexes.
• Detailed methodological settings are provided for the approach.
• The approach was applied to the endogenous human COP9 signalosome complex, revealing its subunit heterogeneity.
• The study highlights the significance of this method for structural mass spectrometry of intact protein complexes.

Conclusions:

• The developed mass spectrometry approach effectively characterizes protein subunit diversity within complexes.
• This method offers a significant advancement for understanding the regulation and function of protein assemblies.
• The findings have implications for structural mass spectrometry and the detailed analysis of protein complexes.