Development of Mass Spectrometry-Screening Methods for Quality Assessment of Cryo-EM Standard

Significance of the Topic

Cryo electron microscopy is a leading tool for structural biology and high-resolution structural determination relies on sample homogeneity and stability. Mass spectrometry screening offers a rapid, sensitive and cost-effective approach for quality control of Cryo-EM standards, enabling improved throughput and more reliable structural outcomes.

Objectives and Study Overview

• Develop native mass spectrometry methods to evaluate sequence integrity of recombinant Apoferritin 24-mer complex
• Assess complex assembly, homogeneity, stability and aggregation as a Cryo-EM standard
• Compare traditional infusion, size-exclusion chromatography MS and online buffer exchange screening workflows
• Correlate MS-derived quality metrics with Cryo-EM grid performance

Methodology and Instrumentation

Recombinant human Apoferritin was expressed in Escherichia coli and purified by glutathione affinity and size-exclusion chromatography. Samples were analyzed by direct infusion, SEC-MS using a Vanquish Flex UHPLC system, and OBE-MS with an online buffer exchange column coupled to a Q Exactive UHMR mass spectrometer. Pseudo MS3 fragmentation was applied for subunit sequence confirmation. Cryo-EM validation was performed on a Titan Krios G4 microscope equipped with a Falcon 4 direct electron detector.

Used Instrumentation

• Thermo Scientific Vanquish Flex UHPLC
• Thermo Scientific Q Exactive UHMR mass spectrometer
• Prototype online buffer exchange column
• Titan Krios G4 Cryo-EM microscope with Falcon 4 detector
• BioPharma Finder and Freestyle software for data analysis

Main Results and Discussion

• Subunit sequence confirmation achieved ~52 percent coverage via pseudo MS3 fragmentation
• SEC-MS and OBE-MS distinguished intact 24-mer assembly from misassembled species with high sensitivity
• OBE-MS workflow reduced analysis time to under five minutes, enabling high-throughput screening
• Aggregation and misfolding percentages increased with storage time and temperature, with samples stored at -80 C and above showing greater degradation
• Native MS quality metrics correlated strongly with Cryo-EM grid particle homogeneity across different purification batches

Benefits and Practical Applications

• Rapid screening of Cryo-EM samples before grid preparation reduces cost and accelerates structure determination
• Data-driven selection of optimal purification strategies and storage conditions
• High-throughput workflows support screening of multiple batches in under five minutes per sample
• Integration of MS and Cryo-EM data streamlines quality control in academic and industrial laboratories

Future Trends and Opportunities

• Further reduction in analysis time via multiplexed native MS and automated data processing
• Integration of machine learning for predictive quality assessment and anomaly detection
• Standardized MS protocols for a broader range of Cryo-EM standards and protein–ligand complexes
• Real-time coupling of MS data with grid preparation for on-the-fly quality control
• Advances in high-resolution MS to resolve subtle post-translational modifications and sample heterogeneity

Conclusion

Native and intact protein mass spectrometry methods including infusion, SEC-MS and online buffer exchange enable comprehensive and rapid quality assessment of Apoferritin Cryo-EM standards. These workflows deliver critical insights into sequence integrity, assembly homogeneity, stability and aggregation that correlate with Cryo-EM grid performance, supporting optimized sample preparation and accelerating structural biology studies.

References

1. Van Aernum P et al Nat Protocols 2020 15 1132 1157
2. Kafader JO et al Nat Methods 2020 17 391 394