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Structural mass spectrometry approaches to study the 20S proteasome
Gili Ben-Nissan1, Shay Vimer1, Mark Tarnavsky1
1Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel.
Methods in Enzymology
|March 27, 2019
Summary
Native mass spectrometry (MS) reveals the intricate structure and composition of the 20S proteasome, a vital protein degradation machine. This technique offers deep insights into the protein complex
Area of Science:
- Biochemistry
- Structural Biology
- Proteomics
Background:
- The 20S proteasome is a universal, large, multisubunit proteolytic machine essential for intracellular protein degradation in archaea and eukaryotes.
- Understanding the 20S proteasome's structure and function is crucial due to its central role in cellular protein homeostasis.
- Previous structural investigations have employed various structural biology methods.
Purpose of the Study:
- To highlight the application of native mass spectrometry (MS) for structural investigations of the 20S proteasome.
- To demonstrate how native MS can reveal the intrinsic heterogeneity, composition, stoichiometry, subunit architecture, and topology of the 20S proteasome.
- To showcase protocols for isolating endogenous 20S proteasomes and analyzing intact complexes and their subunits.
Main Methods:
- Native mass spectrometry (MS) was employed to examine intact 20S proteasome assemblies without disrupting noncovalent interactions.
- Protocols for isolating endogenous 20S proteasomes from yeast, rat liver, and human cells using native MS were described.
- Different instrumental platforms were utilized to analyze the 20S proteasome complex, including its interactions with substrates and regulators.
Main Results:
- Native MS successfully revealed the composition, stoichiometry, and subunit architecture of the 20S proteasome.
- The method provided insights into the intrinsic heterogeneity of isolated 20S proteasome samples.
- Analysis of intact complexes and individual subunits was demonstrated using native MS methodologies.
Conclusions:
- Native MS is a powerful technique for the structural investigation of the 20S proteasome and other large protein assemblies.
- This approach offers a comprehensive understanding of the 20S proteasome's structure, heterogeneity, and interactions.
- Native MS provides valuable insights into the fundamental mechanisms of intracellular protein degradation.
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