Sensitive Native Mass Spectrometry of Macromolecules Using Standard Flow LC/MS

Importance of the Topic

Native mass spectrometry (MS) enables the analysis of intact proteins and complexes under near-physiological conditions, preserving noncovalent interactions. This capability is vital for probing protein–protein interactions, ligand binding, antibody–drug conjugates, and higher-order assemblies with high specificity.

Objectives and Study Overview

This work presents a robust, sensitive workflow for native LC/MS of macromolecules using standard flow rates. It integrates Agilent 1290 Infinity II LC, an AdvanceBio SEC guard column, 6545XT AdvanceBio LC/Q-TOF (extended m/z 30,000), and MassHunter BioConfirm software to achieve reproducible separation, detection, and proteoform analysis of proteins and complexes.

Methodology and Instrumentation

Sample preparation:

• Desalting and buffer exchange to 100 mM ammonium acetate (pH 7) via Bio-Spin P-6 or P-30 cartridges.
• Online SEC separation on AdvanceBio SEC guard column (4.6×30 mm, 200 Å, 1.9 µm) at 0.2 mL/min and 4 °C.
• Injection volume of 1–5 µL of protein solution (0.5–10 mg/mL).

Instrument configuration:

• Agilent 1290 Infinity II LC (high-speed pump, multisampler, column thermostat).
• Agilent 6545XT AdvanceBio LC/Q-TOF with Jet Stream source and large molecule SWARM autotune.
• MassHunter Acquisition and BioConfirm 10.0 for data capture and deconvolution.

Main Results and Discussion

• Myoglobin: Native conditions preserved the holomyoglobin–heme complex (charge states 5+ to 9+), while denaturing solvents led to apomyoglobin and heme dissociation.
• Alcohol dehydrogenase (tetramer, 232 kDa): 100 mM ammonium acetate provided better structural preservation and lower charge states compared to ammonium formate.
• Monoclonal antibodies (NIST mAb and Herceptin): Resolved glycoform envelopes (15+ to 30+ charge states) with low-ppm mass accuracy under native conditions.
• Antibody–drug conjugate T-DM1: Average DAR of 3.5 determined by native MS matched manufacturer specifications.
• Large complexes (pyruvate kinase tetramer, glutamate dehydrogenase hexamer, β-galactosidase tetramer): Detected up to m/z > 10,000 with deconvoluted masses in agreement with expected values.

Benefits and Practical Applications

• Eliminates nanoESI challenges (clogging, aggregation) by using standard LC flow and online desalting.
• Enables accurate characterization of assembly states, proteoforms, and labile noncovalent complexes.
• Provides high reproducibility and throughput for biopharmaceutical development, QC, and structural analysis.

Future Trends and Opportunities

Further automation, integration with ion mobility, and coupling with top-down proteomics will enhance proteoform mapping. Advances in instrumentation are expected to extend mass range, sensitivity, and data analysis for high-throughput screening of large macromolecular assemblies and complex biotherapeutics.

Conclusion

The combined standard-flow SEC and high-performance LC/Q-TOF workflow delivers sensitive, reproducible native MS of intact proteins and complexes. It streamlines preparation, enhances structural preservation, and yields accurate mass and proteoform insights for diverse analytical and biopharmaceutical applications.

References

1. Heck AJ. Native Mass Spectrometry: A Bridge Between Interactomics and Structural Biology. Nat Methods. 2008;5:927–933.
2. Freeke J, Robinson CV, Ruotolo BT. Residual Counter Ions Can Stabilise a Large Protein Complex in the Gas Phase. Int J Mass Spectrom. 2010;298:91–98.
3. Schachner LF, Kelleher NL. Standard Proteoforms and Their Complexes for Native Mass Spectrometry. JASMS. 2019;30:1190–1198.
4. Konermann L, Rosell FL, Mauk AG, Douglas DJ. Acid-Induced Denaturation of Myoglobin Studied by Time-Resolved Electrospray Ionization Mass Spectrometry. Biochemistry. 1997;36(21):6448–6454.
5. Wong DL. Precise Characterization of Intact Monoclonal Antibodies by the Agilent 6545XT AdvanceBio LC/Q-TOF. Agilent application note;5991-7813EN. 2017.