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Published on: November 15, 2017
Using 10,000 Fragment Ions to Inform Scoring in Native Top-down Proteomics
Ashley N Ives1, Taojunfeng Su1, Kenneth R Durbin1,2
1Departments of Chemistry and Molecular Biosciences, the Chemistry of Life Processes Institute, and the Proteomics Center of Excellence, Northwestern University, 2170 Campus Drive, Evanston, Illinois 60208, United States.
New scoring metrics improve native top-down mass spectrometry (nTDMS) by analyzing protein fragmentation patterns. These methods enhance protein identification and characterization, increasing confidence in results for structural mass spectrometry applications.
Area of Science:
- Biochemistry and Proteomics
- Structural Biology
- Mass Spectrometry
Background:
- Protein fragmentation is essential for top-down proteomics, enabling protein identification and proteoform characterization.
- Existing fragmentation analysis methods, like collision-induced dissociation (CID), are well-studied for denatured proteins but show differences for native proteins.
- Native top-down mass spectrometry (nTDMS) requires tailored scoring metrics due to distinct fragmentation behavior in native protein states.
Purpose of the Study:
- To develop and validate new scoring metrics for analyzing protein fragmentation in nTDMS.
- To investigate the relationship between protein topology, residue accessibility, and fragmentation patterns in nTDMS.
- To enhance the accuracy and confidence of protein identification and characterization using nTDMS data.
Main Methods:
- Analysis of 10,252 fragment ions from higher-energy collisional dissociation (HCD) of 159 native monomers and 70 complexes.
- Integration of published structural data to correlate fragmentation events with protein topology and residue solvent accessibility.
- Development of updated C-score and a novel native Fragmentation Propensity Score based on amino acid contribution to fragment ion intensity.
Main Results:
- Fragmentation occurs across a wide range of residue solvent accessibility, indicating complex topological influences.
- Specific amino acid residues, N-terminal aspartic acid and C-terminal proline, significantly contribute to total fragment ion intensity in nTDMS.
- The new scoring systems improved protein identification and characterization, increasing confidence with fewer, high-probability nTDMS fragmentation patterns.
Conclusions:
- The developed scoring metrics effectively enhance the analysis of nTDMS data, addressing the need for native-specific fragmentation analysis.
- These methods provide improved accuracy and confidence for protein identification and characterization in structural mass spectrometry.
- The findings support the increasing utility of nTDMS as a powerful tool for structural mass spectrometry.
