High Throughput Native MS With Robust Ion Source Operation For The Analysis Of Proteins And Protein Complexes

Importance of the Topic

Native mass spectrometry of proteins and their complexes provides detailed structural and compositional information while preserving noncovalent interactions. Streamlining this analysis with higher flow liquid chromatography and a robust ion source enhances throughput and reliability, meeting the demands of proteomics, biopharmaceutical development, and quality control laboratories.

Objectives and Study Overview

This work evaluates the feasibility of applying higher flow LC–MS methods for routine native analysis of proteins and protein complexes. The study focuses on:

• Establishing a stable, high‐throughput ion source workflow.
• Optimizing source parameters to maximize signal intensity.
• Demonstrating preservation of native conformations across multiple protein standards.

Methodology and Instrumentation

Proteins including yeast alcohol dehydrogenase (ADH) tetramer, β‐galactosidase tetramer, and NIST monoclonal antibody were prepared at 20 µM in 200 mM ammonium acetate. Agilent AdvanceBio SEC columns (4.6 × 30 mm, 1.9 µm, 200 Å) were used with a flow rate of 0.1 mL/min on an isocratic pump. After elution, low‐molecular‐weight species were diverted to waste to protect the source.

• Mass spectrometers: 6545XT LC/Q‐TOF and 6560 IM‐QTOF.
• Ion source: Jet Stream with optimized nebulizer (60 psig), nozzle (2000 V), and capillary (5500 V) voltages.
• Sheath and drying gases set to 12 L/min at 400 °C and 12 L/min at 350 °C, respectively.
• Data acquired over m/z ranges up to 14,100 at 0.5 spectra/s using MassHunter and UniDec processing.

Key Results and Discussion

Optimization with ADH tetramer showed that higher sheath and drying gas temperatures and flows produced the strongest signals. Increasing nebulizer pressure further improved ionization efficiency.

• Extended charge state envelopes (e.g., ADH 14+ to 26+) were more pronounced than nanospray benchmarks, suggesting potential conformational differences.
• β‐Galactosidase tetramer and NIST mAb showed minimal denaturation, with intact complex masses matching expected values.
• Ion mobility data revealed multiple conformers for ADH, indicating structural heterogeneity.

Benefits and Practical Applications

This high‐throughput native MS workflow offers:

• Robust, reproducible analysis with a single set of source parameters.
• Six minutes per sample unattended run times supporting large sample batches.
• Preservation of noncovalent assemblies critical for protein‐protein interaction studies and biopharmaceutical characterization.

Future Trends and Applications

Further developments may include:

• Refining ion source parameters tailored to specific protein classes.
• Integrating online buffer exchange for rapid sample cleanup.
• Combining native LC–MS with more advanced ion mobility separations and artificial intelligence–driven data analysis to resolve complex mixtures.
• Expanding applications to membrane proteins, glycoproteins, and large macromolecular assemblies.

Conclusion

This study demonstrates that higher flow LC–MS with a robust ion source can routinely analyze native protein complexes with minimal denaturation. The approach delivers high throughput, consistent performance, and detailed structural insights, making it suitable for research, QC, and biopharmaceutical environments.

Reference

1. VanAernum Z. et al., Rapid Online Buffer Exchange for Native Mass Spectrometry, ChemRxiv (2019).
2. Marty M.T. et al., Bayesian Deconvolution of Mass and Ion Mobility Spectra, Anal. Chem. 87, 4370–4376 (2015).
3. Marty M.T., Eliminating Artifacts in Electrospray Deconvolution, J. Am. Soc. Mass Spectrom. 30, 2174–2177 (2019).
4. Schachner L.F. et al., Standard Proteoforms for Native MS, J. Am. Soc. Mass Spectrom. 30, 1190–1198 (2019).
5. Raj S.B. et al., Yeast Alcohol Dehydrogenase Structure and Catalysis, Biochemistry 53, 5791–5805 (2014).