Reassembling protein complexes after controlled disassembly by top-down mass spectrometry in native mode
Luis F Schachner1, Denise P Tran1, Alexander Lee1
1Departments of Chemistry, Chemical and Biological Engineering, and Molecular Biosciences, the Chemistry of Life Processes Institute, and the Proteomics Center of Excellence, Northwestern University, 2170 Tech Dr., Silverman Hall, 60208, Evanston, IL, USA.
International Journal of Mass Spectrometry
|September 20, 2021
Summary
Native top-down mass spectrometry (nTDMS) offers deep insights into protein complexes. This tutorial presents a decision-tree to standardize the analysis of nTDMS fragmentation data for improved proteoform characterization.
Area of Science:
- Structural Biology
- Proteomics
- Mass Spectrometry
Background:
- Native top-down mass spectrometry (nTDMS) combines electrospray ionization in native mode with top-down mass spectrometry.
- nTDMS provides intact mass, subunit masses, cofactor information, and fragment ion data in a single experiment.
- Analysis of fragmentation data for proteoform characterization in nTDMS is not yet standardized.
Purpose of the Study:
- To provide a decision-tree for analyzing nTDMS experiments on protein complexes and bioassemblies.
- To offer strategies and highlight software for interrogating fragment ions in nTDMS data.
- To guide researchers in designing nTDMS experiments and data processing.
Main Methods:
- Utilized electrospray ionization in native mode coupled with top-down mass spectrometry (nTDMS).
- Developed a decision-tree framework for analyzing nTDMS fragmentation data.
- Included example datasets and highlighted software for hypothesis-driven interrogation of fragment ions.
Main Results:
- Demonstrated the capability of nTDMS to provide multi-level structural information on proteins and complexes.
- Presented a systematic approach to navigate and analyze complex nTDMS fragmentation data.
- Highlighted key features for experimental and data processing design, including deconvolution, search mode, and validation.
Conclusions:
- Standardized analysis of nTDMS fragmentation data is crucial for comprehensive proteoform characterization.
- The proposed decision-tree facilitates hypothesis-driven interrogation of PTMs, metals, and cofactors on native proteoforms.
- Guidance on software selection (open-access and commercial) aids in optimizing nTDMS experimental and data processing design.
