Top-Down Analysis of Branched Proteins Using Mass Spectrometry

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Post-translational modifications (PTMs) involving small signaling proteins like Rub1 (NEDD8) and ubiquitin are crucial for protein function and fate.
• Current analytical methods for studying these modifications, particularly branched protein structures, are limited, scarce, and labor-intensive.

Purpose of the Study:

• To develop and validate a novel, efficient strategy for analyzing branched proteins using top-down mass spectrometry.
• To enable the identification of participating proteins, define conjugation sites, and characterize other modifications in branched protein systems.

Main Methods:

• Top-down mass spectrometry utilizing electron-transfer dissociation (ETD) with collisional activation.
• Development of specialized software for graphic interpretation of mass spectrometry data.
• Application to recombinant proteins and synthetic peptides modified with ubiquitin or Rub1.

Main Results:

• Successfully identified and characterized four recombinant branched proteins and one synthetic peptide.
• The novel strategy accurately identified constituent proteins, defined conjugation sites, and characterized additional modifications.
• Demonstrated the ability to elucidate structures of unknown, two-component branched proteins.

Conclusions:

• The developed top-down mass spectrometry approach provides a powerful and direct method for analyzing branched proteins.
• This strategy overcomes limitations of existing methods, offering high resolution and accuracy for PTM characterization.
• The approach holds potential for extension to more complex branched protein systems and modifications.