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Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies
Published on: November 28, 2017
The impact of protein characterization in structural proteomics
Arie Geerlof1, J Brown, B Coutard
1EMBL Hamburg Outstation, Notkestrasse 85, Hamburg, Germany. arie.geerlof@embl-hamburg.de
Acta Crystallographica. Section D, Biological Crystallography
|September 27, 2006
Summary
This study introduces a protein characterization strategy for structural proteomics, emphasizing quality assessment before crystallization. It recommends purity thresholds to improve protein production and crystallization success rates.
Area of Science:
- Structural proteomics
- Biochemistry
- Molecular biology
Background:
- Protein characterization is crucial for structural proteomics, impacting protein preparation quality and structure determination.
- Poor protein quality is a major bottleneck in obtaining well-diffracting crystals for structural studies.
- Assessing protein preparations before crystallization is essential for evaluating the production process.
Purpose of the Study:
- To propose a protein production and crystallization strategy incorporating quality assessment.
- To define threshold values for protein purity and monodispersity to guide optimization.
- To describe applications and developments in protein characterization methods for structural proteomics.
Main Methods:
- Mass spectrometry (MS)
- Fluorescence spectroscopy
- Static light scattering (SLS)
- Analytical ultracentrifugation (AUC)
- Small-angle X-ray scattering (SAXS)
Main Results:
- A strategy is proposed with recommended thresholds: protein purity (95%) and monodispersity (85%).
- Below these thresholds, further optimization of protein production is strongly advised.
- The paper details the application of various characterization methods within the EC Structural Proteomics in Europe contract.
Conclusions:
- Implementing rigorous protein characterization improves the success rate of structural proteomics pipelines.
- Defined quality control metrics (purity, monodispersity) are vital for efficient protein production and crystallization.
- Advanced biophysical techniques are essential for comprehensive protein characterization, including interactions and modifications.
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