Rapid online buffer exchange - Solutions for high-throughput analysis of large biomolecules by native mass spectrometry

Importance of the Topic

Native mass spectrometry (nMS) preserves non-covalent assemblies and delivers rapid, high-sensitivity structural information on large biomolecules. However, non-volatile buffers commonly used in protein production and purification interfere with MS analysis. Traditional offline buffer exchange is laborious and limits throughput. Rapid online buffer exchange (OBE) nMS overcomes these challenges by coupling size-exclusion separation with native MS, enabling automated, high-throughput screening and quality control within minutes.

Objectives and Study Overview

This technical note describes the principles and practical implementation of OBE nMS for large biomolecule analysis. It aims to:

• Explain the separation mechanism of online buffer exchange.
• Detail multiple LC configurations for coupling OBE to MS.
• Demonstrate rapid desalting and structural screening of proteins and complexes.
• Provide method recommendations for flow rates, buffers and valve timing.

Methodology and Instrumentation

OBE nMS uses short size-exclusion columns to separate proteins (>8 kDa) from salts and small contaminants. Aqueous mobile phases of 50–200 mM ammonium acetate maintain native structures and minimize analyte–stationary phase interactions. Key setups include:

• Dual-pump Vanquish Flex UHPLC with 2-position/6-port switching valve.
• Single-pump Vanquish with syringe drive and optional divert valve.
• UltiMate 3000 RSLCnano with micro-flow selector and divert valve.

Online valve switching is scheduled to load the sample in a low-residence column mode (Position LOAD) and then divert non-volatiles to waste (Position WASTE) while preserving the protein signal. Typical isocratic flows are 50–100 µL/min. MS acquisition on the Q Exactive UHMR employs extended trapping and HCD for intact mass detection within m/z 1,000–14,000.

Major Results and Discussion

Using a NativePac OBE-1 column (2.1×50 mm, 80 Å), 1 µg of NIST mAb or BSA in PBS was buffer-exchanged into 200 mM ammonium acetate in under 5 minutes. Chromatograms show clean separation of the void-volume protein peak from later salt elution. Mass spectra of the exchanged mAb revealed its glycoform distribution without salt adducts, confirming efficient desalting and structural integrity. Consistent elution times across protein sizes simplify method transfer and automation.

Benefits and Practical Applications

OBE nMS provides:

• High-throughput quality control of expressed proteins and complexes.
• Automated desalting compatible with MS-labile interactions.
• Workflow guidance for expression and purification optimization.
• Rapid screening of cell lysates or partially purified samples.

This approach accelerates decision making in biopharma R&D, structural biology and QA/QC laboratories.

Future Trends and Opportunities

Advances in column materials and microfluidic integration will further reduce dead volumes and sample consumption. Coupling OBE nMS with ion-mobility separation and real-time data analysis will expand its value in complex sample characterization. Integration into automated platforms promises fully hands-free native proteoform screening and high-throughput biomarker validation.

Conclusion

Rapid online buffer exchange enhances native MS workflows by automating desalting and enabling fast, sensitive structural profiling of large biomolecules. The flexibility of LC configurations and robust separation performance support diverse applications in research and industry. OBE nMS is a powerful tool for accelerating protein analysis and streamlining quality control in modern analytical laboratories.

Used Instrumentation

• Thermo Scientific Vanquish Flex UHPLC System
• Thermo Scientific UltiMate 3000 RSLCnano System
• Thermo Scientific NativePac OBE-1 SEC column, 2.1×50 mm, 80 Å, 3 µm
• Thermo Scientific Q Exactive UHMR mass spectrometer
• Biocompatible Viper and nanoViper capillaries and 2-position/6-port switching valves

References

1. VanAernum ZL, Busch F, Jones BJ, Jia M, Chen Z, Boyken SE, Sahasrabuddhe A, Baker D, Wysocki VH. Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry. Nature Protocols. 2020;15:1132–1157.
2. Liu W, Zhang T, Bechler S, Viner R. Rapid online buffer exchange for protein screening. Thermo Fisher Scientific Application Note FL0170. 2022.